DNA encoding hyaluronan synthase from Pasteurella multocida and methods of use

ABSTRACT

The present invention relates to a nucleic acid segment having a coding region segment encoding enzymatically active  Pasteurella multocida  hyaluronate synthase (PmHAS), and to the use of this nucleic acid segment in the preparation of recombinant cells which produce hyaluronate synthase and its hyaluronic acid product.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/217,613, filedAug. 12, 2002; which is a continuation of U.S. Ser. No. 09/283,402,filed Apr. 1, 1999, now abandoned; which claims benefit under 35 U.S.C.119(e) of U.S. Ser. No. 60/080,414, filed on Apr. 2, 1998; each of whichis hereby expressly incorporated by reference herein in its entirety.Said application U.S. Ser. No. 10/217,613 is also a continuation-in-partof U.S. Ser. No. 09/178,851, filed Oct. 26, 1998, now abandoned; whichis hereby expressly incorporated by reference herein in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This application was supported in part by a National Research Initiativegrant for Sustaining Animal Health and Well-Being 94-37204-0929 from theU.S. Department of Agriculture. The United States Government may haverights in and to this application by virtue of this funding.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a DNA sequence encoding hyaluronansynthase from Pasteurella multocida. More particularly, the presentinvention relates to a DNA sequence encoding hyaluronan synthase fromPasteurella multocida which is capable of being placed into arecombinant construct so as to be able to express hyaluronan synthase ina foreign host. The present invention also relates to methods of using aDNA sequence encoding hyaluronan synthase from Pasteurella multocida to(1) make hyaluronan polymers of varying size distribution; (2) makehyaluronan polymers incorporating substitute or additional base sugars;(3) develop new and novel animal vaccines; and (4) develop new and noveldiagnostic tests for the detection and identification of animalpathogens.

2. Brief Description of the Background Art

The polysaccharide hyaluronic acid (“HA”) or hyaluronan is an essentialcomponent of higher animals that serves both structural and recognitionroles. In mammals and birds, HA is present in large quantities in theskin, the joint synovial fluid, and the vitreous humor of the eye.Certain pathogenic bacteria, namely, Gram-positive Group A and CStreptococcus and Gram-negative Pasteurella multocida Carter Type A,produce extracellular capsules containing HA with the same chemicalstructure as the HA molecule found in their vertebrate hosts. This“molecular mimicry” foils attempts to mount a strong antibody responseto the capsular polysaccharide. In contrast, capsular polysaccharideswith different structures produced by other bacteria are often quiteantigenic. The HA capsule also apparently helps the pathogens evade hostdefenses including phagocytosis.

Historically, researchers in the field have not succeeded in cloning oridentifying Hyaluronan Synthase (“HAS”) from Pasteurella. Bacterial HASenzymes from Group A & C Streptococcus have been identified and cloned.HasA from Streptococcus pyogenes was the first HAS to be definitivelyidentified. This integral membrane protein utilizes intracellularUDP-GlcA and UDP-GlcNAc as substrates. The nascent HA chain is extrudedthrough the membrane to form the extracellular capsule. A Xenopusprotein, DG42, has also been determined to be a HAS. Several human andmurine homologs of DG42, named HAS1, HAS2 and HAS3, have also beenidentified. There is considerable similarity among these molecularlycloned mammalian enzymes at the amino acid level, but they reside ondifferent chromosomes. The unique HAS from P. multocida has a primarystructure that does not strongly resemble the previously cloned enzymesfrom Streptococcus, PBCV-1 virus or higher animals.

A viral HAS, with an ORF called A98R, has been identified as being28-30% identical to the streptococcal and vertebrate enzymes. PBCV-1(Paramecium bursaria Chlorella virus) produces an authentic HApolysaccharide shortly after infection of its Chlorella-like green algaehost. A98R is the first virally encoded enzyme identified as producing acarbohydrate polymer.

Carter type A P. multocida, the causative agent of fowl cholera, isresponsible for great economic losses in the U.S. poultry industry.Acapsular mutants of P. multocida do not thrive in the bloodstream ofturkeys after intravenous injection, where encapsulated parental strainsmultiply quickly and cause death within 1 to 2 days. Spontaneouslyarising mutant strain which is acapsular, was also 10⁵-fold lessvirulent than wild-type, but the nature of the genetic defects in allthe cases before the disclosed mutant (as described hereinafter) was notknown.

Pasteurella bacterial pathogens cause extensive losses to U.S.agriculture. The extracellular polysaccharide capsule of P. multocidahas been proposed to be a major virulence factor. The Type A capsule iscomposed of a polysaccharide, namely HA, that is identical to the normalpolysaccharide in the host's body and thus invisible to the immunesystem. This “molecular mimicry” also hinders host defenses such asphagocytosis and complement-mediated lysis. Furthermore, HA is notstrongly immunogenic since the polymer is a normal component of the hostbody. The capsules of other bacteria that are composed of differentpolysaccharides, however, are usually major targets of the immuneresponse. The antibodies generated against capsular polymers are oftenresponsible for clearance of microorganisms and long-term immunity.

Knowing the factors responsible for a pathogen's virulence providesclues on how to defeat the disease intelligently and efficaciously. InType A P. multocida, one of the virulence factors is the protectiveshield of nonimmunogenic HA, an almost insurmountable barrier for hostdefenses. A few strains do not appear to rely on the HA capsule forprotection, but utilize other unknown factors to resist the hostmechanisms. Alternatively, these strains may possess much smallercapsules that are not detected by classical tests.

For chickens and especially turkeys, fowl cholera can be devastating. Afew to 1,000 cells of some encapsulated strains can kill a turkey in24-48 hours. Fowl cholera is an economically important disease in NorthAmerica. Studies done in the late 1980s show some of the effects of fowlcholera on the turkey industry: (i) fowl cholera causes 14.7 to 18% ofall sickness, (ii) in one state alone the annual loss was $600,000,(iii) it costs $0.40/bird to treat a sick flock with antibiotics, and(iv) it costs $0.12/bird for treatment to prevent infection.

Certain strains of Type A P. multocida cause pneumonic lesions andshipping fever in cattle subjected to stress. The subsequent reductionin weight gain at the feedlot causes major losses. The bovine strainsare somewhat distinct from fowl cholera strains, but the molecular basisfor these differences in host range preference is not yet clear. Type Aalso causes half of the pneumonia in swine. Type D P. multocida is mostwell known for its involvement in atrophic rhinitis, a high prioritydisease in swine.

Type D capsular polymer has an unknown structure that appears to be sometype of glycosaminoglycan; this is the same family of polymers thatincludes HA. This disease is also precipitated by Bordetellabronchiseptica, but the condition is worse when both bacterial speciesare present. It is estimated that Type F causes about 10% of the fowlcholera caes. In this case, the capsular polymer is not HA, but arelated polymer called chondroitin.

Currently, disease prevention on the fowl range is mediated by twoelements: vaccines and antibiotics, as well as strict sanitation. Theutility of the first option is limited, since there are many serotypesin the field and vaccines are only effective against a limited subset ofthe entire pathogen spectrum. Killed-cell vaccine is dispensed bylabor-intensive injection, and the protection obtained is not high.Therefore, this route is usually reserved for the breeder animals. Moreeffective live-cell vaccines can be delivered via the water supply, butit is difficult to dose a flock of thousands evenly. Additionally, live“avirulent” vaccines can sometimes cause disease themselves if the birdsare otherwise stressed or sick. The most common reason for thisunpredictability is that these avirulent strains arose from spontaneousmutations in unknown or uncharacterized genes. Protocols that utilizerepeated alternating exposure to live and dead vaccines can protectbirds only against challenge with the same serotype.

The second disease prevention option is antibiotics. These are used ateither subtherapeutic doses to prevent infection or at high doses tocombat fowl cholera in infected birds. The percentage of birds withdisease may drop with drug treatment, but timely and extensive treatmentis necessary. Late doses or premature withdrawal of antibiotics oftenresults in chronic fowl cholera and sickly birds with abscesses orlesions that lead to condemnation and lost sales. Furthermore, sinceresistant strains of P. multocida continually arise and drug costs arehigh, this solution is not attractive in the long run. In addition, TypeF P. multocida may cause 5-10% of fowl cholera in North America. Avaccine directed against Type A strains may not fully protect againstthis other capsular type if it emerges as a major pathogen in thefuture. In the cattle and swine industries, no vaccine has been totallysatisfactory. Prophylactic antibiotic treatment is used to avoid lossesin weight gain, but this option is expensive and subject to themicrobial resistance issue.

In the present invention, enzymes involved in making the protectivebacterial HA capsule have been identified at the gene/DNA level. Theidentification of these enzymes will lead to disease intervention byblocking capsule synthesis of pathogens with specific inhibitors thatspare host HA biosynthesis. For example, a drug mimicking the substratesused to make HA or a regulator of the P. multocida HA synthase stopsproduction of the bacterial HA polysaccharide, and thus blocks capsuleformation. This is a direct analogy to many current antibiotics thathave dissimilar effects on microbial and host systems. This approach ispreferred because the P. multocida HA synthase and the vertebrate HAsynthase are very different at the protein level. Therefore, it islikely that the enzymes also differ in reaction mechanism or substratebinding sites.

P. multocida, once stripped of its protective capsule shield issignificantly more vulnerable a target for host defenses. Phagocytesreadily engulfed and destroyed by the acapsular microbes. The hostcomplement complex reaches and disrupts the sensitive outer membrane ofbacteria. Antibodies are more readily generated against the newlyexposed immunogens, such as the lipopolysaccharides and surface proteinsthat determine somatic serotype in P. multocida. These antibodies arebetter able to bind to acapsular cells later in the immune response.Thus, the immune response from vaccinations are more effective and morecost-effective. Capsule-inhibiting drugs are substantial additions tothe treatment of fowl cholera.

The present invention and use of the capsule biosynthesis of Type A P.multocida aids in the understanding of the other capsular serotypes. DNAprobes have been used to type A capsule genes to establish that Type Dand F possess similar homologs.

High molecular weight HA also has a wide variety of usefulapplications—ranging from cosmetics to eye surgery. Due to its potentialfor high viscosity and its high biocompatibility, HA finds particularapplication in eye surgery as a replacement for vitreous fluid. HA hasalso been used to treat racehorses for traumatic arthritis byintra-articular injections of HA, in shaving cream as a lubricant, andin a variety of cosmetic products due to its physiochemical propertiesof high viscosity and its ability to retain moisture for long periods oftime. In fact, in August of 1997 the U.S. Food and Drug Agency approvedthe use of high molecular weight HA in the treatment of severe arthritisthrough the injection of such high molecular weight HA directly into theaffected joints. In general, the higher molecular weight HA that isemployed the better. This is because HA solution viscosity increaseswith the average molecular weight of the individual HA polymer moleculesin the solution. Unfortunately, very high molecular weight HA, such asthat ranging up to 10⁷, has been difficult to obtain by currentlyavailable isolation procedures.

To address these or other difficulties, there is a need for new methodsand constructs that can be used to produce HA having one or moreimproved properties such as greater purity or ease of preparation. Inparticular, there is a need to develop methodology for the production oflarger amounts of relatively high molecular weight and relatively pureHA than is currently commercially available. There is yet another needto be able to develop methodology for the production of HA having amodified size distribution (HA_(Δsize)) as well as HA having a modifiedstructure (HA_(Δmod)).

The present invention, therefore, functionally characterizes the Type AP. multocida genes involved in capsule biosynthesis, assesses the roleof the capsule as a virulence factor in fowl cholera, and has obtainedthe homologous genes involved in Type D and F capsule biosynthesis. Withthis information, vaccines have been developed utilizing “knock out” P.multocida genes that do not produce HAS. These acapsular avirulentstrains have the ability to act as vaccines for fowl cholera or shippingfever.

SUMMARY OF THE INVENTION

The present invention relates to a novel HAS that produces HA. Usingvarious molecular biology techniques, a gene for a new HAS was found infowl cholera pathogen Type A Pasteurella multocida. This new HAS fromPasteurella multocida, (“PmHAS”), was cloned and shown to be functionalin other species of bacteria.

Thus, a new source of HA has been identified. The DNA sequence of PmHASmay also be used to generate potential attenuated vaccine strains of P.multocida bacteria after knocking out the normal microbial gene byhomologous recombination with a disrupted version. Additionally, thePmHAS DNA sequence allows for the generation of diagnostic bacterialtyping probes for related P. multocida types that are agriculturalpathogens of fowl, cattle, sheep and swine.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a partial sequence alignment of PmHAS P. multocida and otherglycosyltransferases from other bacteria.

FIG. 2 is a sequence alignment of residues 342-383 of PmHAS as comparedto residues 362-404 of the mammalian UDP-GalNAc:polypeptideGalNAc-transferase.

FIG. 3 is an autoradiogram representation of a photoaffinity labelingstudy with UDP-sugar analogs of PmHAS.

FIG. 4 is an autoradiogram depicting the reduced or absent photaffinitylabeling of PmHAS in various Tn mutants of PmHAS.

FIG. 5 depicts photomicrographs demonstrating HA production inrecombinant E. coli.

FIG. 6 graphically depicts the construction of pPmHAS and its subcloninginto an expression vector.

FIG. 7 depicts the pH dependence of PmHAS activity.

FIG. 8 depicts metal dependence of HAS activity.

FIG. 9 depicts HAS activity dependence on UDP-GlcNAc concentration.

FIG. 10 depicts HAS activity dependence on UDP-GlcA concentration.

FIG. 11 is a Hanes-Woolf plot estimation of V_(MAX) and K_(m).

FIG. 12 is a Southern blot mapping of Tn mutants.

FIG. 13 depicts chimeric DNA templates for sequence analysis of Tndisruption sites.

FIG. 14 is a diagrammatic representation of a portion of the HAbiosynthesis locus of Type A P. multocida.

FIG. 15 is a Southern blot analysis of various capsule types of P.multocida with Type A capsule gene probes.

FIG. 16 is an electrophoretogram of the PCR of the Type A DNA andheterologous DNA with various Type A primers.

FIG. 17 is a partial sequence comparison of type A and F KfaA homologsand E. coli KfaA.

FIG. 18 is a schematic of wild-type HAS gene versus a knockout mutantgene.

FIG. 19 is the molecular biological confirmation of the acapsularknockout mutant by Southern blot and PCR analyses.

FIG. 20 is a sequence comparison of Type A and F P. multocida.

FIG. 21 is a Western blot anaylsis of native and recombinant PmHASproteins.

DETAILED DESCRIPTION OF THE INVENTION

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or illustrated in the drawings. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

As used herein, the term “nucleic acid segment” and “DNA segment” areused interchangeably and refer to a DNA molecule which has been isolatedfree of total genomic DNA of a particular species. Therefore, a“purified” DNA or nucleic acid segment as used herein, refers to a DNAsegment which contains a Hyaluronate Synthase (“HAS”) coding sequenceyet is isolated away from, or purified free from, unrelated genomic DNA,for example, total Pasteurella multocida or, for example, mammalian hostgenomic DNA. Included within the term “DNA segment”, are DNA segmentsand smaller fragments of such segments, and also recombinant vectors,including, for example, plasmids, cosmids, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified PmHAS generefers to a DNA segment including HAS coding sequences isolatedsubstantially away from other naturally occurring genes or proteinencoding sequences. In this respect, the term “gene” is used forsimplicity to refer to a functional protein, polypeptide or peptideencoding unit. As will be understood by those in the art, thisfunctional term includes genomic sequences, cDNA sequences orcombinations thereof. “Isolated substantially away from other codingsequences” means that the gene of interest, in this case PmHAS, formsthe significant part of the coding region of the DNA segment, and thatthe DNA segment does not contain large portions of naturally-occurringcoding DNA, such as large chromosomal fragments or other functionalgenes or DNA coding regions. Of course, this refers to the DNA segmentas originally isolated, and does not exclude genes or coding regionslater added to, or intentionally left in the segment by the hand of man.

Due to certain advantages associated with the use of prokaryoticsources, one will likely realize the most advantages upon isolation ofthe HAS gene from the prokaryote P. multocida. One such advantage isthat, typically, eukaryotic enzymes may require significantpost-translational modifications that can only be achieved in aeukaryotic host. This will tend to limit the applicability of anyeukaryotic HA synthase gene that is obtained. Moreover, those ofordinary skill in the art will likely realize additional advantages interms of time and ease of genetic manipulation where a prokaryoticenzyme gene is sought to be employed. These additional advantagesinclude (a) the ease of isolation of a prokaryotic gene because of therelatively small size of the genome and, therefore, the reduced amountof screening of the corresponding genomic library, and (b) the ease ofmanipulation because the overall size of the coding region of aprokaryotic gene is significantly smaller due to the absence of introns.Furthermore, if the product of the PmHAS gene (i.e., the enzyme)requires posttranslational modifications, these would best be achievedin a similar prokaryotic cellular environment (host) from which the genewas derived.

Preferably, DNA sequences in accordance with the present invention willfurther include genetic control regions which allow the expression ofthe sequence in a selected recombinant host. Of course, the nature ofthe control region employed will generally vary depending on theparticular use (e.g., cloning host) envisioned.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences which encode a PmHASgene, that includes within its amino acid sequence an amino acidsequence in accordance with SEQ ID NO:1. Moreover, in other particularembodiments, the invention concerns isolated DNA segments andrecombinant vectors incorporating DNA sequences which encode a gene thatincludes within its amino acid sequence the amino acid sequence of anHAS gene or DNA, and in particular to an HAS gene or cDNA, correspondingto Pasteurella multocida HAS. For example, where the DNA segment orvector encodes a full length HAS protein, or is intended for use inexpressing the HAS protein, preferred sequences are those which areessentially as set forth in SEQ ID NO:1

Truncated PmHAS also falls within the definition of preferred sequencesas set forth in SEQ ID NO:1. For instance, at the c terminus,approximately 270-272 amino acids may be removed from the sequence andstill have a functioning HAS (SEQ ID NO:17). Those of ordinary skill inthe art would appreciate that simple amino acid removal from either endof the PmHAS sequence can be accomplished. The truncated versions of thesequence simply have to be checked for HAS activity in order todetermine if such a truncated sequence is still capable of producingHAS.

Nucleic acid segments having HA synthase activity may be isolated by themethods described herein. The term “a sequence essentially as set forthin SEQ ID NO:1 means that the sequence substantially corresponds to aportion of SEQ ID NO:1 and has relatively few amino acids which are notidentical to, or a biologically functional equivalent of, the aminoacids of SEQ ID NO:1. The term “biologically functional equivalent” iswell understood in the art and is further defined in detail herein, as agene having a sequence essentially as set forth in SEQ ID NO:1, and thatis associated with the ability of prokaryotes to produce HA or ahyaluronic acid coat.

The art is replete with examples of practitioners ability to makestructural changes to a nucleic acid segment (i.e., encoding conservedor semi-conserved amino acid substitutions) and still preserve itsenzymatic or functional activity. See for example: (1) Risler et al.“Amino Acid Substitutions in Structurally Related Proteins. A PatternRecognition Approach.” J. Mol. Biol. 204:1019-1029 (1988) [“ . . .according to the observed exchangeability of amino acid side chains,only four groups could be delineated; (i) Ile and Val; (ii) Leu and Met,(iii) Lys, Arg, and Gln, and (iv) Tyr and Phe.”]; (2) Niefind et al.“Amino Acid Similarity Coefficients for Protein Modeling and SequenceAlignment Derived from Main-Chain Folding Anoles.”]. Mol. Biol.219:481-497 (1991) [similarity parameters allow amino acid substitutionsto be designed]; and, (3) Overington et al. “Environment-Specific AminoAcid Substitution Tables: Tertiary Templates and Prediction of ProteinFolds,” Protein Science 1:216-226 (1992) [“Analysis of the pattern ofobserved substitutions as a function of local environment shows thatthere are distinct patterns . . . ” Compatible changes can be made.]

These references and countless others, indicate that one of ordinaryskill in the art, given a nucleic acid sequence, could makesubstitutions and changes to the nucleic acid sequence without changingits functionality. Also, a substituted nucleic acid segment may behighly identical and retain its enzymatic activity with regard to itsunadulterated parent, and yet still fail to hybridize thereto.

The invention discloses nucleic acid segments encoding an enzymaticallyactive hyaluronate synthase from P. multocida—PmHAS. One of ordinaryskill in the art would appreciate that substitutions can be made to thePmHAS nucleic acid segment listed in SEQ ID NO:2 without deviatingoutside the scope and claims of the present invention. Standardized andaccepted functionally equivalent amino acid substitutions are presentedin Table A. TABLE A Conservative and Semi- Amino Acid Group ConservativeSubstitutions NonPolar R Groups Alanine, Valine, Leucine, Isoleucine,Proline, Methionine, Phenylalanine, Tryptophan Polar, but uncharged, RGroups Glycine, Serine, Threonine, Cysteine, Asparagine, GlutamineNegatively Charged R Groups Aspartic Acid, Glutamic Acid PositivelyCharged R Groups Lysine, Arginine, Histidine

Another preferred embodiment of the present invention is a purifiednucleic acid segment that encodes a protein in accordance with SEQ IDNO:1, further defined as a recombinant vector. As used herein, the term“recombinant vector” refers to a vector that has been modified tocontain a nucleic acid segment that encodes an HAS protein, or fragmentthereof. The recombinant vector may be further defined as an expressionvector comprising a promoter operatively linked to said HAS encodingnucleic acid segment.

A further preferred embodiment of the present invention is a host cell,made recombinant with a recombinant vector comprising an HAS gene. Thepreferred recombinant host cell may be a prokaryotic cell. In anotherembodiment, the recombinant host cell is a eukaryotic cell. As usedherein, the term “engineered” or “recombinant” cell is intended to referto a cell into which a recombinant gene, such as a gene encoding HAS,has been introduced. Therefore, engineered cells are distinguishablefrom naturally occurring cells which do not contain a recombinantlyintroduced gene. Engineered cells are thus cells having a gene or genesintroduced through the hand of man. Recombinantly introduced genes willeither be in the form of a cDNA gene, a copy of a genomic gene, or willinclude genes positioned adjacent to a promoter not naturally associatedwith the particular introduced gene.

In preferred embodiments, the HA synthase-encoding DNA segments furtherinclude DNA sequences, known in the art functionally as origins ofreplication or “replicons”, which allow replication of contiguoussequences by the particular host. Such origins allow the preparation ofextrachromosomally localized and replicating chimeric segments orplasmids, to which HA synthase DNA sequences are ligated. In morepreferred instances, the employed origin is one capable of replicationin bacterial hosts suitable for biotechnology applications. However, formore versatility of cloned DNA segments, it may be desirable toalternatively or even additionally employ origins recognized by otherhost systems whose use is contemplated (such as in a shuttle vector).

The isolation and use of other replication origins such as the SV40,polyoma or bovine papilloma virus origins, which may be employed forcloning or expression in a number of higher organisms, are well known tothose of ordinary skill in the art. In certain embodiments, theinvention may thus be defined in terms of a recombinant transformationvector which includes the HA synthase coding gene sequence together withan appropriate replication origin and under the control of selectedcontrol regions.

Thus, it will be appreciated by those of skill in the art that othermeans may be used to obtain the HAS gene or cDNA, in light of thepresent disclosure. For example, polymerase chain reaction or RT-PCRproduced DNA fragments may be obtained which contain full complements ofgenes or cDNAs from a number of sources, including other strains ofPasteurellas or from eukaryotic sources, such as cDNA libraries.Virtually any molecular cloning approach may be employed for thegeneration of DNA fragments in accordance with the present invention.Thus, the only limitation generally on the particular method employedfor DNA isolation is that the isolated nucleic acids should encode abiologically functional equivalent HA synthase.

Once the DNA has been isolated it is ligated together with a selectedvector. Virtually any cloning vector can be employed to realizeadvantages in accordance with the invention. Typical useful vectorsinclude plasmids and phages for use in prokaryotic organisms and evenviral vectors for use in eukaryotic organisms. Examples includepKK223-3, pSA3, recombinant lambda, SV40, polyoma, adenovirus, bovinepapilloma virus and retroviruses. However, it is believed thatparticular advantages will ultimately be realized where vectors capableof replication in both Lactococcus or Bacillus strains and E. coli areemployed.

Vectors such as these, exemplified by the pSA3 vector of Dao andFerretti or the pAT19 vector of Trieu-Cuot, et al., allow one to performclonal colony selection in an easily manipulated host such as E. coli,followed by subsequent transfer back into a food grade Lactococcus orBacillus strain for production of HA. These are benign and well studiedorganisms used in the production of certain foods and biotechnologyproducts. These are advantageous in that one can augment the Lactococcusor Bacillus strain's ability to synthesize HA through gene dosaging(i.e., providing extra copies of the HA synthase gene by amplification)and/or inclusion of additional genes to increase the availability of HAprecursors. The inherent ability of a bacterium to synthesize HA canalso be augmented through the formation of extra copies, oramplification, of the plasmid that carries the HA synthase gene. Thisamplification can account for up to a 10-fold increase in plasmid copynumber and, therefore, the HA synthase gene copy number.

Another procedure that would further augment HA synthase gene copynumber is the insertion of multiple copies of the gene into the plasmid.Another technique would include integrating the HAS gene intochromosomal DNA. This extra amplification would be especially feasible,since the bacterial HA synthase gene size is small. In some scenarios,the chromosomal DNA-ligated vector is employed to transfect the hostthat is selected for clonal screening purposes such as E. coli, throughthe use of a vector that is capable of expressing the inserted DNA inthe chosen host.

In certain other embodiments, the invention concerns isolated DNAsegments and recombinant vectors that include within their sequence anucleic acid sequence essentially as set forth in SEQ ID NO:2 The term“essentially as set forth in SEQ ID NO:2 is used in the same sense asdescribed above and means that the nucleic acid sequence substantiallycorresponds to a portion of SEQ ID NO:2 and has relatively few codonswhich are not identical, or functionally equivalent, to the codons ofSEQ ID NO:2 The term “functionally equivalent codon” is used herein torefer to codons that encode the same amino acid, such as the six codonsfor arginine or serine, as set forth in Table A, and also refers tocodons that encode biologically equivalent amino acids.

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N— or C-terminalamino acids or 5′ or 3′ nucleic acid sequences, and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein activity where protein expression andenzyme activity is concerned. The addition of terminal sequencesparticularly applies to nucleic acid sequences which may, for example,include various non-coding sequences flanking either of the 5′ or 3′portions of the coding region or may include various internal sequences,which are known to occur within genes. Furthermore, residues may beremoved from the N or C terminal amino acids and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, as well.

Allowing for the degeneracy of the genetic code as well as conserved andsemi-conserved substitutions, sequences which have between about 40% andabout 80%; or more preferably, between about 80% and about 90%; or evenmore preferably, between about 90% and about 99%; of nucleotides whichare identical to the nucleotides of SEQ ID NO:2 will be sequences whichare “essentially as set forth in SEQ ID NO:2”. Sequences which areessentially the same as those set forth in SEQ ID NO:2 may also befunctionally defined as sequences which are capable of hybridizing to anucleic acid segment containing the complement of SEQ ID NO:2 understandard or less stringent hybridizing conditions. Suitable standardhybridization conditions will be well known to those of skill in the artand are clearly set forth herein.

The term “standard hybridization conditions” as used herein, is used todescribe those conditions under which substantially complementarynucleic acid segments will form standard Watson-Crick base-pairing. Anumber of factors are known that determine the specificity of binding orhybridization, such as pH, temperature, salt concentration, the presenceof agents, such as formamide and dimethyl sulfoxide, the length of thesegments that are hybridizing, and the like. When it is contemplatedthat shorter nucleic acid segments will be used for hybridization, forexample fragments between about 14 and about 100 nucleotides, salt andtemperature preferred conditions for hybridization will include1.2-1.8×HPB at 40-50° C.

Naturally, the present invention also encompasses DNA segments which arecomplementary, or essentially complementary, to the sequence set forthin SEQ ID NO:2. Nucleic acid sequences which are “complementary” arethose which are capable of base-pairing according to the standardWatson-Crick complementarity rules. As used herein, the term“complementary sequences” means nucleic acid sequences which aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of SEQ ID NO:2.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, epitope tags, polyhistidine regions, other coding segments, and the like, such that theiroverall length may vary considerably. It is therefore contemplated thata nucleic acid fragment of almost any length may be employed, with thetotal length preferably being limited by the ease of preparation and usein the intended recombinant DNA protocol.

Naturally, it will also be understood that this invention is not limitedto the particular amino acid and nucleic acid sequences of SEQ ID NO:1and 2. Recombinant vectors and isolated DNA segments may thereforevariously include the HAS coding regions themselves, coding regionsbearing selected alterations or modifications in the basic codingregion, or they may encode larger polypeptides which neverthelessinclude HAS-coding regions or may encode biologically functionalequivalent proteins or peptides which have variant amino acidssequences.

The DNA segments of the present invention encompass biologicallyfunctional equivalent HAS proteins and peptides. Such sequences mayarise as a consequence of codon redundancy and functional equivalencywhich are known to occur naturally within nucleic acid sequences and theproteins thus encoded. Alternatively, functionally equivalent proteinsor peptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the enzyme activity or to antigenicity of the HASprotein or to test HAS mutants in order to examine HA synthase activityat the molecular level.

Also, specific changes to the HAS coding sequence can result in theproduction of HA having a modified size distribution or structuralconfiguration. One of ordinary skill in the art would appreciate thatthe HAS coding sequence can be manipulated in a manner to produce analtered hyaluronate synthase which in turn is capable of producinghyaluronic acid having differing polymer sizes and/or functionalcapabilities. For example, the HAS coding sequence may be altered insuch a manner that the hyaluronate synthase has an altered sugarsubstrate specificity so that the hyaluronate synthase creates a newhyaluronic acid-like polymer incorporating a different structure such asa previously unincorporated sugar or sugar derivative. This newlyincorporated sugar could result in a modified hyaluronic acid havingdifferent functional properties, a hyaluronic acid having a smaller orlarger polymer size/molecular weight, or both. As will be appreciated byone of ordinary skill in the art given the HAS coding sequences, changesand/or substitutions can be made to the HAS coding sequence such thatthese desired property and/or size modifications can be accomplished.

The term “modified structure” as used herein denotes a hyaluronic acidpolymer containing a sugar or derivative not normally found in thenaturally occurring HA polysaccharide. The term “modified sizedistribution” refer to the synthesis of hyaluronic acid molecules of asize distribution not normally found with the native enzyme; theengineered size could be much smaller or larger than normal.

Various hyaluronic acid products of differing size have application inthe areas of drug delivery and the generation of an enzyme of alteredstructure can be combined with a hyaluronic acid of differing size.Applications in angiogenesis and wound healing are potentially large ifhyaluronic acid polymers of about 20 monosaccharides can be made in goodquantities. Another particular application for small hyaluronic acidoligosaccharides is in the stabilization of recombinant human proteinsused for medical purposes. A major problem with such proteins is theirclearance from the blood and a short biological half life. One presentsolution to this problem is to couple a small molecule shield thatprevents the protein from being cleared from the circulation toorapidly. Very small molecular weight hyaluronic acid is well suited forthis role and would be nonimmunogenic and biocompatible. Largermolecular weight hyaluronic acid attached to a drug or protein may beused to target the reticuloendothelial cell system which has endocyticreceptors for hyaluronic acid.

One of ordinary skill in the art given this disclosure would appreciatethat there are several ways in which the size distribution of thehyaluronic acid polymer made by the hyaluronate synthase could beregulated to give different sizes. First, the kinetic control of productsize can be altered by decreasing temperature, decreasing time of enzymeaction and by decreasing the concentration of one or both sugarnucleotide substrates. Decreasing any or all of these variables willgive lower amounts and smaller sizes of hyaluronic acid product. Thedisadvantages of these approaches are that the yield of product willalso be decreased and it may be difficult to achieve reproducibilityfrom day to day or batch to batch.

Secondly, the alteration of the intrinsic ability of the enzyme tosynthesize a large hyaluronic acid product. Changes to the protein canbe engineered by recombinant DNA technology, including substitution,deletion and addition of specific amino acids (or even the introductionof prosthetic groups through metabolic processing). Such changes thatresult in an intrinsically slower enzyme could then allow morereproducible control of hyaluronic acid size by kinetic means. The finalhyaluronic acid size distribution is determined by certaincharacteristics of the enzyme, that rely on particular amino acids inthe sequence. Among the 20% of residues absolutely conserved between thestreptococcal enzymes and the eukaryotic hyaluronate synthases, there isa set of amino acids at unique positions that control or greatlyinfluence the size of the hyaluronic acid polymer that the enzyme canmake. Specific changes in any of these residues can produce a modifiedHAS that produces an HA product having a modified size distribution.Engineered changes to seHAS, spHAS, pmHAS, or cvHAS that decrease theintrinsic size of the hyaluronic acid that the enzyme can make beforethe hyaluronic acid is released, will provide powerful means to producehyaluronic acid product of smaller or potentially larger size than thenative enzyme.

Finally, larger molecular weight hyaluronic acid made be degraded withspecific hyaluronidases to make lower molecular weight hyaluronic acid.This practice, however, is very difficult to achieve reproducibility andone must meticulously repurify the hyaluronic acid to remove thehyaluronidase and unwanted digestion products.

Structurally modified hyaluronic acid is no different conceptually thanaltering the size distribution of the hyaluronic acid product bychanging particular amino acids in the desired HAS or the spHAS.Derivatives of UDP-GlcNAc, in which the N-acetyl group is missing(UDP-GlcN) or replaced with another chemically useful group, areexpected to be particularly useful. The strong substrate specificitymust rely on a particular subset of amino acids among the 20% that areconserved. Specific changes to one or more of these residues creates afunctional synthase that interacts less specifically with one or more ofthe substrates than the native enzyme. This altered enzyme could thenutilize alternate natural or special sugar nucleotides to incorporatesugar derivatives designed to allow different chemistries to be employedfor the following purposes: (i) covalently coupling specific drugs,proteins, or toxins to the structurally modified hyaluronic acid forgeneral or targeted drug delivery, radiological procedures, etc. (ii)covalently cross linking the hyaluronic acid itself or to other supportsto achieve a gel, or other three dimensional biomaterial with strongerphysical properties, and (iii) covalently linking hyaluronic acid to asurface to create a biocompatible film or monolayer.

The present invention relates to a novel HAS that produces HA. Usingvarious molecular biology techniques, a gene for a new HAS was found infowl cholera pathogen Type A Pasteurella multocida. This new HAS fromPasteurella multocida, or PmHAS, has been cloned and shown to befunctional in other species of bacteria. The PmHAS protein polymerizesauthentic HA polysaccharide.

The carbohydrate produced by a recombinant E. coli transformed withPmHAS is recognized by the cartilage HA-binding protein and is sensitiveto HA lyase digestion. Both of these reagents are regarded by those ofordinary skill in the art as being specific for HA polysaccharide. Also,both UDP-GlcA and UDP-GlcNAc were required for HA synthesis in vitro.Azido-UDP-GlcA and azido-UDP-GlcNAc, but not azido-UDP-Glc, specificallyphotoincorporated into PmHAS. As in the case of streptococcal HasA andXenopus DG42, it appears that one polypeptide species, PmHAS, transferstwo distinct sugar groups to the nascent HA chain.

Many encapsulated Gram-negative bacteria, including E. coli, Neisseriameningitidis, and Hemophilus influenzae, possess clusters of genesresponsible for capsule biosynthesis organized in operons. These operonsoften contain genes encoding (i) enzymes required for sugar nucleotideprecursor synthesis, (ii) glycosyltransferases for polymerizing theexopolysaccharide, and (iii) proteins implicated in polysaccharideexport. The Type A P. multocida HA capsule operon contains (i) a KfaAanalog, (ii) a HA synthase, and (iii) a putative UDP-Glc dehydrogenase.The Tn916 elements in the P. multocida acapsular mutants H and L werenot integrated directly in the HAS gene but rather were located in theKfaA homolog gene.

As the PmHAS exists in a locus of at least several genes essential formaking polysaccharide, a lesion or defect in any one of the capsulegenes could affect HA production and capsule formation in Pasteurella.Thus, by disrupting an adjacent gene a vaccine could also be made. Forexample, if UDP-Glc dehydrogenase is removed or disrupted, no precursorsugar for HA synthase is available and HA cannot be made. Also, if Kfaor another transport associated gene is killed, then no surface HA ismade by the microbe. Thus, the product of HA synthase in the naturalPasteurella microbe, i.e., an HA capsule, could be stopped by (a)disrupting precursor formation, or (b) disrupting the polymerizationmachinery, or (c) disrupting the transport machinery.

At the amino acid level, PmHAS is not as similar to the other clonedHASs as one of ordinary skill in the art would expect. Two potentialshort motifs, DGS(S/T) (SEQ ID NO:18) at residues 477-480 and DSD atresidues 527-529 of PmHAS are present in HasA. Another similarDGS-containing motif is found repeated at residues 196-198 of PmHAS. TheDG of the first motif and the DSD are conserved in all HASs. However,several absolutely conserved motifs ((S/G)GPLXXY (SEQ ID NO:19),GDDRXLTN (SEQ ID NO:20), and LXQQXRWXKS(Y/F/W)(F/C)RE) (SEQ ID NO:21)found in all previously cloned HASs are absent from PmHAS. Instead, avariety of bacterial glycosyltransferases align more closely with thesequence in the central portion of the P. multocida HAS protein. Theseenzymes, which have been either shown or predicted to transfer GlcNAc,galactose, or GalNAc groups, are roughly one-third the size of the PmHASand their amino acid termini sequences align together with the middle ofthe PmHAS polypeptide, residues 430-540.

Sections of the first 420 residues of PmHAS show some similarity toportions of the mammalian UDP-GalNAc:polypeptide GalNAc-transferase.These observations may be a reflection of a possible domain structurewithin PmHAS. The last approximate 340 residues of the PmHAS are notsignificantly similar to other entries in the sequence data bases.Therefore, the P. multocida HAS is unique and is most likely theprototype of an entire new class of HAS.

PmHAS is roughly twice the size of the streptococcal, viral, orvertebrate HASs—972 versus 417-588 residues, respectively. Furthermore,the hydropathy plots of PmHAS and the other known HASs are dissimilar.Utilizing the TMPRED program, which is readily known and available tothose of ordinary skill in the art on the World Wide Web, PmHAS ispredicted to have only two candidate transmembrane helices (centered onresidues 170 and 510), and both termini of the protein may be located inthe cytoplasm. Topologically, these assumptions imply that one-third ofthe P. multocida polypeptide (approximately 340 residues) is locatedoutside of the cytoplasm. On the other hand, a different topology ispredicted for the other classes of HAS.

Reporter enzyme fusion analysis of streptococcal HAS confirms that adifferent topological arrangement exists in this enzyme consisting of(i) two transmembrane helices near the amino acid terminus, (ii) aputative cytoplasmic domain, followed by (iii) three membrane-associatedregions at the carboxyl half of the protein. The connecting loopsbetween membrane-associated regions are rather short (4-10 residues);therefore, the vast majority of the polypeptide chain is probably notextracellularly exposed.

The following detailed experimental steps and discussion of results,confirms that the present invention relates to a novel and unique PmHAS.

1. Molecular Cloning of PmHAS

Tn916 insertional mutagenesis and probe generation was first completed.Tn916 was used to disrupt and to tag the P. multocida HA biosynthesislocus. The Tn element on a nonreplicating plasmid, pAM150 was introducedinto a wild-type encapsulated P. multocida strain (ATCC number 15742) byelectroporation. Altered colony morphology was initially screened byvisual examination with oblique lighting. The Wild-type strain formslarge mucoid (“wet” appearance) colonies that appear iridescent (red andgreen coloration). Smaller, “drier” colonies lacking iridescence werechosen and streaked out. India ink staining and light microscopy wereused as a secondary screen to assess the state of encapsulation. Theposition of the Tn elements in the mutant chromosome was mapped bySouthern analysis.

The DNA sequences at the Tn-disrupted sites from several independentlyselected mutants were obtained by direct dideoxy sequence analysis oftagged chromosomal DNA. Briefly, a chimeric DNA fragment consisting of a12-kb portion of the Tn916 element and a short region of the P.multocida DNA generated by HhaI digestion of mutant chromosomal DNA waspurified by agarose gel electrophoresis (all of the wild type HhaIgenomic fragments are less than or equal to 7 kb). The chimeric fragmentserved as the template in cycle sequencing reactions using ³³Pterminators and a Tn916 right arm terminus primer(5′-GACCTTGATAAAGTGTGATAAGTCC-3′) (SEQ ID NO:22). The sequence data wereused to design PCR primers. Gel-purified PCR products were labeled withdigoxigenin utilizing the High Prime system manufactured by BoehringerMannheim and well known to those of ordinary skill in the art.

The next step was the isolation of a functional HAS locus. A λ libraryof Sau3A partially digested wild type DNA was made using BamHI-cleavedλZap Express vector system produced by Stratagene. The plaque lifts werescreened by hybridization with digoxigenin-labeled PCR product.Escherichia coli XLI-Blue MRF′ was co-infected with individual purifiedpositive λ clones and ExAssist helper phage to yield phagemids. Theresulting phagemids were transfected into E. coli XLOLR cells to recoverthe plasmids.

The plasmids were transformed into a host more suitable for HApolysaccharide production, E. coli K5 (strain Bi8337-41). This strainproduces UDP-GlcA, a required substrate for HA biosynthesis that is notfound at significant levels in most laboratory strains. Additionally, K5possesses many other genes essential for capsular polysaccharidetransport in E. coli. Another host employed for expression studies wasE. coli EV5, an acapsular derivative of a K1 strain which produces apolysialic acid capsule and which also possesses all the same generalcapsular polysaccharide transport machinery as K5, but does not havehigh levels of UDP-Glc dehydrogenase.

Cultures of the E. coli transformants with the candidate plasmids grownin completely defined medium were tested for HA polysaccharideproduction as described previously except that the cell pellets wereextracted with 8 M urea, 0.01% SDS at 95 degrees Celsius for 2 minutes.The HA test assay produced by Pharmacia Biotech Inc., which is wellknown by those of ordinary skill in the art, employs a specificHA-binding protein to detect HA at concentrations greater than or equalto 0.1 μg/ml. Multiple determinations of HA levels were averaged. The HAconcentration in bacterial cultures was normalized for differences incell number by measuring the A₆₀₀ value and presenting the data as μgHA/ml/A₆₀₀ of bacteria. One plasmid, pPm7A, with a 5.8 kb insertconferred E. coli K5 with the ability to produce HA; no HA was producedby cells with vector plasmid alone. A truncated derivative of pPm7Acontaining an approximately 3.3 kb insert, called pPmΔ6e, could directthe biosynthesis of HA when transformed into E. coli K5. Therefore, thesequence of both strands of the pPm7A plasmid corresponding to thepPmΔ6e DNA was determined. A single complete 972-residue ORF, which wecalled PmHAS, was found and is shown in SEQ ID NO:1. The correspondingnucleotide sequence is shown in SEQ ID NO:2.

Expression of recombinant P. multocida HAS was then undertaken. ThePmHAS ORF in the pPm7A insert was amplified by 13 cycles of PCR with Taqpolymerase and primers corresponding to the sequence near the deducedamino and carboxyl termini (codons in capital letters: sense,5′-gcgaattcaaaggacagaaaATGAAcACATTATCACAAG-3′ (SEQ ID NO:23), andantisense, 5′-gggaattctgcagttaTAGAGTTATACTATTAATAATGAAC-3′ (SEQ IDNO:24); start and stop codons, respectively, in bold). Codon 2 (T→C) wasaltered (italic lowercase letter) to increase protein production in E.coli. The primers also contained EcoRI and PstI restriction sites(underlined letters) to facilitate cloning into the expression plasmidpKK223-3 (tac promoter; Pharmacia). The resulting recombinant construct,pPmHAS, was transformed into E. coli SURE cells (Stratagene) and thisstrain was used as the source of membrane preparations for in vitro HASassays. Log phase cultures(LB broth, 30 degrees Celsius) were inducedwith 0.5 mM isopropylthiogalactoside for 3 hours before harvest. Theplasmid was also transformed into E. coli K5; the resulting strain wasexamined for the presence of capsule by light microscopy and buoyantdensity centrifugation. The K5 bacterial cultures were not inducedroutinely because isopropylthiogalactopyranoside addition did notincrease HA levels in LB or defined medium significantly.

Photoaffinity labeling of the native P. multocida HAS was thenundertaken. The radiolabeled UDP sugar analogs, [³²P]azido-UDP-GlcA (3mCi/μMol) and [³²P]azido-UDP-GlcNAc (2.5 mCi/μMol), were prepared andpurified as described in the literature and known to one of ordinaryskill in the art. Membrane preparations from P. multocida wild type in50 mM Tris, 20 mM MgCl₂, pH 7, were incubated with either probe (finalconcentration, 20 μM) for 30 seconds on ice before irradiation withultraviolet light (254 nm, 90 seconds). The proteins were precipitatedwith 5% trichloroacetic acid before SDS-polyacrylamide gelelectrophoresis analysis. No radiolabel was incorporated if theirradiation step was omitted. As a specificity control, 10-fold molarexcess of the normal UDP sugar was co-incubated with the probe andmembranes. [³²P]azido-UDP-Glc (3 mCi/μMol) was also used as anothercontrol.

Approximately 8×10⁴ Tn-containing transformants produced by severalrounds of mutagenesis were screened for differences in colonymorphology. By light spectroscopy with India Ink, the cells from smallnoniridescent colonies (n=4) possessed no detectable capsule(acapsular), whereas the cells from medium-sized iridescent colonies(n=8) appeared to have a capsule of about 10-25% of the diameter of thewild type (microcapsular). Two of the acapsular mutants, named H and L,which had Tn elements that mapped to the same HindIII or BstXI genomicfragments reverted to wild-type colony morphology at rates ofapproximately 10⁻³. The Tn element in each revertant had excised fromthe original position and reinserted at different, new locations asjudged by Southern analysis; on the other hand, all acapsular subclonesretained the Tn element at the original location. No significant HASactivity was detected in membrane preparations from mutant H cells,whereas substantial HAS activity was obtained from wild-type wild typecells (less than or equal to 0.7 versus 120 pmol transfer GlcA/mgprotein/h, respectively). These findings suggest that the Tn elements inmutants H and L were indeed responsible for disrupting the HAbiosynthesis locus.

In order to bridge the gap between the Tn insertion sites of twoacapsular mutants, PCR using the mutant L chromosomal DNA template wasperformed with a primer derived from sequence at the mutant H disruptionsite, PmHF (5′-CTCCAGCTGTAAATTAGAGATAAAG-3′) (SEQ ID NO:25), and aprimer corresponding to the left terminus of Tn916, TnL2(5′-GCACATAGAATAAGGCTTTACGAGC-3′) (SEQ ID NO:26). A specificapproximately 1 kb PCR product was obtained; alternatively, no productwas formed if PmHR (inverse complement of PmHF) or the Tn916 right armprimer were substituted. The PCR product was used as a hybridizationprobe to obtain a functional copy of the P. multocida HAS.

Six positively hybridizing plaques were found after screeningapproximately 10⁴ plaques, and these phage were converted into plasmids.One plasmid, pPm7A, was found that could direct E. coli K5 to produce HAin vivo (20 μg HA/ml/A₆₀₀ of bacteria). E. coli K5 with control plasmidsdid not produce HA (less than or equal to 0.05 μg HA/ml/A₆₀₀). E. coliXLOLR or E. coli EV5 cells (which lack UDP-Glc dehydrogenase activity)do not produce HA even if they contain the pPm7A plasmid (less than orequal to 0.05 μg HA/ml/A₆₀₀). This genetic evidence implies that theinsert of pPm7A does not encode a functional UDP-Glc dehydrogenaseenzyme.

A truncated derivative of the pPm7A plasmid with the smallest insertcapable of directing HA biosynthesis (85 μg HA/ml/A₆₀₀ of K5 bacteria),pPmΔ6e, contained a single complete ORF (SEQ ID NO:2) encoding a 972residue protein as shown in SEQ ID NO:1. No obvious promoter is presentin SEQ ID NO:2, but there is a predicted ribosome binding site labeledin bold “centered on nucleotides −10 to −7 and the two putativetransmembrane regions predicted by TMPRED are underlined (Residues162-182, and 503-522). The PmHAS of SEQ ID NO:1 is twice as large asstreptococcal HasA. This protein is the HA synthase from P. multocida,PmHAS. The predicted M_(r) is 111,923 and the calculated isoelectricpoint is 6.84. SEQ ID NO:2 is the nucleotide sequence for PmHAS.

This PmHAS was used as the query in BLASTP searches of the proteinsequence data base. The central portion of PmHAS (residues 436-536) ismost homologous to bacterial glycosyltransferases from a wide variety ofgenera, including Streptococcus, Vibrio, Neisseria, and Staphylococcus,that form exopolysaccharides or the carbohydrate portions oflipopolysaccharides (smallest sum probabilities, 10⁻²²-10⁻¹⁰, as shownin FIG. 1. FIG. 1 graphically depicts the sequence alignment of P.multocida HAS and other glycosyltransferases. The MULTALIN alignmentillustrates that the central region of the PmHAS (residues 436-536) ismost similar to the amino-terminal portions of various enzymes thatproduce other exopolysaccharides (Streptococcus thermophilus EpsI; Type14 S. pneumoniae Cps14J) or the carbohydrate moiety oflipopolysaccharides (H. Influenzae LgtD homology). Only a few of thepossible examples are shown in FIG. 1. S. pyogenes HasA (residues61-168) has limited similarity to this depicted region of PmHAS.

The most notable sequence similarities are the DGSTD (SEQ ID NO:27) andDXDD (SEQ ID NO:28) motifs. Unexpectedly, there was no significantoverall similarity of PmHAS to the streptococcal, viral, or vertebrateHASs with HASA having the smallest sum probability of 0.33. Only oneshort region of streptococcal HasA aligns with PmHAS in a convincingmanner and is shown in FIG. 1.

A few segments of the first half of PmHAS are also similar to portionsof the mammalian UDP-GalNAc:polypeptide GalNAc-transferase, an enzymethat initiates O-glycosylation of mucin-type proteins with the smallestsum probability being approximately 10⁻³, FIG. 2. As shown in FIG. 2,the sequence alignment of residues 342-383 of PmHAS are most similar toresidues 362-404 of the mammalian UDP-GalNAc:polypeptideGalNAc-transferase. For both FIGS. 1 and 2, the identical residues arebold and underlined, and the consensus symbols are: !, either I or V; #,any one of N, D, E, or Q; %, either F or Y. The clusters of acidicresidues are well conserved throughout the sequences.

The partial ORF (27 residues) downstream of PmHAS is very similar to theamino terminus of several UDP-Glc dehydrogenases from bacteria includingE. coli, Salmonella typhimurium, and Streptococcus pneumoniae (67-74%identity). The severe truncation in the original pPm7A clone would beexpected to result in complete loss of dehydrogenase activity. The otherORF (623 residues) upstream of PmHAS is very homologous to the E. coliK5 KfaA protein with a smallest sum probability of 10⁻⁵², a proteinputatively involved in the transport of capsular polysaccharide out ofthe cell.

The predicted size of 972 residues (112 kDa) for PmHAS was confirmed byphotoaffinity labeling of membrane preparations from P. multocida wildtype. Both [³²P]azido-UDP-GlcA and [³²P]azido-UDP-GlcNAc probesphotoincorporated into an approximately 110 kDa protein in anUV-dependent manner. FIG. 3 is a photoaffinity labeling of the PmHASwith UDP-sugar analogs. [³²P]azido-UDP-GlcA and [³²P]azido-UDP-GlcNAcwere incubated with membrane preparations (45 μg of protein) isolatedfrom wild-type P. multocida and irradiated with UV light. Autoradiograms(5 day exposures) of 10% SDS-PAGE gels are shown in FIG. 3. Both probesphotolabel an approximately 110 kDa protein in an UV-dependent manner(the “−” lanes). In order to assess the specificity ofphotoincorporation, a parallel sample was treated identically exceptthat the reaction mixtures included a 10-fold excess of unlabeledcompetitor (UDP-GlcNAc or UDP-GlcA, respectively; marked the “+” lanes).The band intensities are reduced in comparison to the “−” lanes. Thestandards are marked in kDa.

Competition with the corresponding unlabeled natural UDP-sugarprecursors lowered the extent of probe photoincorporation. In parallelexperiments, [³²P]azido-UDP-Glc, an analog of the normal HA precursors,did not label this 110 kDa protein. Furthermore, membranes derived fromTn mutants had either no or very low amounts of azido-UDP-GlcAphotoincorporation into this protein. As shown in FIG. 4, membranepreparations (60 μg of protein) from wild-type (W) or various acapsularTn mutants (A, G, or H) were photolabeled with [³²P]azido-UDP-GlcA. Theregion of the autoradiogram in the vicinity of the approximate 110 kDaprotein is shown in FIG. 4. No photoincorporation is seen in the A and Gsamples. The small extent of photolabelling in the H sample is due tothe low rate of reversion observed with this particular mutant. The sizeof the photoaffinity labeled protein in the W sample corresponds well tothe predicted M_(r) of the cloned PmHAS ORF.

Membranes derived from E. coli SURE cells containing the pPmHAS plasmid,but not samples from cells with the vector pKK223-3 alone, synthesizedHA in vitro when supplied with both UDP-GlcA and UDP-GlcNAc (25 versusless than or equal to 1.5 pMol GlcA transfer/mg protein/hour,respectively). No incorporation of [¹⁴C]GlcA was observed if UDP-GlcNAcwas omitted or if divalent metal ions were chelated with EDTA. The HASactivity derived from recombinant HAS was similar to the enzyme obtainedfrom wild-type P. multocida membranes because Mn²⁺ stimulated at least10-fold more activity than Mg²⁺.

Cultures of recombinant E. coli were also tested for the presence of HApolysaccharide with a radiometric assay utilizing labeled HA-bindingprotein. E. coli K5 with pPmHAS produced 460 μg HA/ml/A₆₀₀. K5 cellswith pKK223-3 vector alone did not produce HA (less than or equal to0.05 μg HA/ml/A₆₀₀. For comparison, wild-type P. multocida wild typegrown in the same media produced 1,100 μg HA/ml/A₆₀₀ . E. coli K5 withpPmHAS produced such high levels of HA that the cells becameencapsulated. As shown in FIG. 5, Panel A, the photomicrographs ofrecombinant E. coli with India ink staining (1,000× magnification)reveals that E. coli K5 cells with pPmHAS produce a substantial capsulethat appears as a white halo around the cells.

The radius of the capsule of the recombinant strain was approximately0.2-0.5 μm (assuming a bacterial cell width of 0.5 μm). This capsulecould be removed by treatment with either bovine testicularhyaluronidase or Streptomyces HA lyase. As shown in FIG. 5, Panel B, thecapsular material was removed from the E. coli K5(pPmHAS) cells by brieftreatment with Streptomyces HA lyase. Thus, PmHAS directs polymerizationof the HA polysaccharide.

Neither the native K5 host strain nor transformants containing pKK223-3vector possessed a readily observable capsule as determined by lightspectroscopy. K5 cells with pPmHAS were also deemed encapsulated bybuoyant density centrifugation. The recombinant cells floated on top ofthe 58% Percoll cushion, whereas the vector control cells orhyaluronidase-treated recombinant cells pelleted through the Percollcushion.

The p/PmHAS plasmid in E. coli K5 is the first generation system formaking recombinant HA with PmHAS; other optimized vectors and/or hostsmay give greater yields, and these other optimized vectors and/or hostsare herein contemplated for use with the present invention. One ofordinary skill in the art, given this disclosure, would be capable ofoptimizing such vectors and/or hosts.

2. Enzymological Characterization of PmHAS

Protein was determined by the Coomassie dye-binding assay utilizing abovine serum albumin standard. P. multocida wild type (American TypeCulture Collection 15742), a highly virulent turkey strain that formsvery mucoid colonies, was maintained on brain/heart infusion mediumunder aerobic conditions at 37 degrees Celsius. An acapsular mutant ofthe strain which formed smaller, “drier” colonies, named TnA, wasgenerated by a newly described Tn916 insertational mutagenesis methoddescribed herein.

Total membranes from P. multocida were prepared by a modification of themethod for producing HA synthase from E. coli with recombinant plasmidscontaining hasA. Cells were grown with vigorous shaking to mid-log phase(0.4-0.8 A₆₀₀) and then bovine testicular hyaluronidase (Sigma Type V,20 units/mL final) was added to remove the capsule. After 40 min., thecells were chilled on ice and harvested by centrifugation (2000×g for 15min). The cells were washed twice PBS by repeated suspension andcentrifugation, and the cell pellet could be stored at −80 degreesCelsius. All of the following steps were performed on ice unless notedotherwise.

The cells were resuspended by pipetting in 1/400 the original culturevolume of 20% sucrose and 30 mM Tris, pH 8.0, containing the proteaseinhibitors pepstatin and leupeptin. Cell lysis was carried out by usinglysozyme digestion (addition of 1/10 the suspended volume of 4 mg/mLenzyme in 0.1 M EDTA, 40-min incubation) followed by ultrasonicdisruption (power setting 3, three cycles of 30 s on/off; Heat SystemsW-380 with microprobe). Before the ultrasonification step, sodiumthioglycolate was added to the mixture (0.1 mM final concentration)following the addition of phenylmethanesulfonyl fluoride. In all theremaining manipulations, the PBS also contained freshly addedthioglycolate at the same concentration.

The lysate was treated with DNase and RNase (1 μg/mL each, 10 min at 4degrees Celsius) and the cellular debris was removed by low-speedcentrifugation (10000×g for 1 hour). The supernatant fraction wasdiluted 6-fold with PBS and the membrane fraction was harvested byultracentrifugation (100000×g for 1 hour). The pellet was washed twiceby repeated suspension in PBS containing 10 mM MgCl₂ followed byultracentrifugation. For generating membrane preparations used in metalspecificity studies, MgCl₂ was omitted and replaced with 0.2 mM EDTAduring the wash steps. Membrane preparations were suspended in 50 mMTris, pH 7, and 0.1 mM thioglycolate, at a concentration of 1-3 mg/mLprotein and stored at −80 degrees Celsius.

HA synthase activity was routinely detected by incorporation of theradiolabel derived from the sugar nucleotide precursor UDP-[¹⁴C]GlcA(0.27 Ci/mmol, ICN), into higher molecular weight products. The variousassay buffers, described in the figure legends, also contained 0.3 mMDTT. Assays (100 μL final volume were initiated by addition of membranesto the reaction mixture and incubation at 37 degrees Celsius. After 1hour, the reactions were terminated by addition of SDS (2% final) andmixing. For the kinetic studies, the product and precursors wereseparated by descending paper chromatography (Whatman 3M with 65:35ethanol/1 M ammonium acetate, pH 5.5). The HA polysaccharide at theorigin of the paper chromatogram was eluted with water before liquidscintillation counting. The assays were typically performed underconditions in which no more than 5% of the precursors were consumed bylimiting amounts of enzyme.

Controls to verify incorporation into authentic HA included omission ofthe required second sugar nucleotide precursor or digestion using thespecific hyaluronidase from Streptomyces hyalurolyticus. Gel-filtrationchromatography with SEPHACRYL™ S-200 (Pharmacia) in PBS was used toassess the molecular weight of the radiolabeled polymer formed in vitrounder optimized assay conditions. These samples were treated as forpaper chromatography except that, after termination, they were heated at95 degrees Celsius for 2 minutes and clarified by centrifugation(15000×g for 7 minutes) before application to the column.

EDTA (0.2 mM) was used to chelate any metal ions present in assaymixtures to verify metal dependence of the HAS activity. Variousdivalent metals, including Mg, Mn, Cu, Co, and Ni, were tested as theirchloride salts. The K_(m) values of the substrates were estimated bytitration of one sugar nucleotide concentration while holding the otherradiolabeled precursor at a constant and saturating concentration. Forthese studies, UDP-[³H]GlcNAc (30 Ci/mmol, NEN) was employed as well asthe UDP-[¹⁴C]GlcA precursor.

P. multocida cells produce a readily visible extracellular HA capsule,and since the streptococcal HasA is a transmembrane protein, membranepreparations of the fowl cholera pathogen were tested. In early trials,crude membrane fractions derived from ultrasonication alone possessedvery low levels if UDP-GlcNAc-dependent UDP-[⁴C]GlcA incorporating intoHA [approximately 0.2 pmol of GlcA transfer (μg of proteins)⁻¹h⁻¹] whenassayed under conditions similar to those for measuring streptococcalHAS activity. The enzyme from E. coil with the recombinant hasA plasmidwas also recalcitrant to isolation at first. These results were incontrast to the easily detectable amounts obtained from Streptococcus bysimilar methods.

An alternative preparation protocol using ice-cold lysozyme treatment inthe presence of protease inhibitors in conjunction with ultrasonicationallowed the substantial recovery of HAS activity from both species ofGram-negative bacteria. Specific activities of 5-10 pmol of GlcAtransfer (μg of protein)⁻¹h⁻¹ were routinely obtained for crudemembranes of wild-type P. multocida with the new method. In the absenceof UDP-GlcNAc, virtually no radioactivity (less than 1% of identicalassay with both sugar precursors) from UDP-[¹⁴C]GlcA was incorporatedinto higher molecular weight material. Membranes prepared from theacapsular mutant, TnA, posssessed no detectable HAS activity whensupplemented with both sugar nucleotide precursors. Gel-filtrationanalysis using a Sephacryl S-200 column indicates that the molecularmass of the majority of the ¹⁴C-labeled product synthesized in vitro is≧8×10⁴ Da since the material elutes in the void volume; such a valuecorresponds to a HA molecule composed of at least 400 monomers. Thisproduct is also sensitive to Streptomyces hyaluronidase digestion butresistant to Pronase treatment.

The parameters of the HAS assay were varied to maximize incorporation ofUDP-sugars into polysaccharide by P. multocida membranes. StreptococcalHasA requires Mg²⁺ and therefore this metal ion was included in theinitial assays of P. multocida membranes. The P. multocida HAS wasrelatively active from pH 6.5 to 8.6 in Tris-type buffers with anoptimum at pH 7, FIG. 7. FIG. 7 depicts the pH dependence of P.multocida HAS activity. The incorporation of [¹⁴C]GlcA into HApolysaccharide catalyzed by membranes (38 μg of protein) was measured inreactions buffered at various pH values (50 mMTris/2-(N-(morpholino)ethanesulfonic acid, bis-Tris/HCl, or tris/Hcl; nomajor buffer ion-specific effects were noted). The incubation mixturealso contained 20 mM MgCl₂, 120 μM UDP-GlcA (4.5×10⁴ dpm/assay), and 300μM UDP-GlcNAc. The incorporation of the assay using the optimal buffer,pH 7 Tris, was set to 100% activity. A broad pH optimum aroundneutrality was observed.

The HAS activity was linear with respect to the incubation time atneutral pH for at least 1 hour. The P. multocida enzyme was apparentlyless active at higher ionic strengths because the addition of 100 mMNaCl to the reaction containing 50 mM Tris, pH 7, and 20 mM MgCl₂reduced sugar incorporation by approximately 50%.

The metal ion specificity of the P. multocida HAS was assessed at pH 7,FIG. 8. FIG. 8 depicts metal dependence of HAS activity. The productionof HA was measured in the presence of increasing concentrations of Mg(circles) or Mn (squares) ion. The membranes (46 μg of protein),prewashed with 0.2 mM EDTA were incubated in a mixture of the metal ionin 50 mM Tris, pH 7, 120 μM UDP-GlcA (4.5×10⁴ dpm/assay), and 300 μMUDP-GlcNAc for 1 hour. The background with no metal present (22 dpm) wassubtracted from each point. Mn is more effective than Mg.

Under metal-free conditions in the presence of EDTA, no incorporation ofradiolabeled precursor into polysaccharide was detectable (<0.5% ofmaximal signal). Mn²⁺ gave the highest incorporation rates at the lowestion concentrations for the tested metals (mg, Mn, Co, Cu, and Ni). Mg²⁺gave about 50% of the Mn²⁺ stimulation but at 10-fold higherconcentrations. Co²⁺ or Ni²⁺ at 10 mM supported lower levels of activity(20% or 9%, respectively, of 1 mM Mn²⁺ assays), but membranes suppliedwith 10 mM Cu²⁺ were inactive. Indeed, mixing 10 mM Cu²⁺ and 20 mM Mg²⁺with the membrane preparation resulted in almost no incorporation oflabel into polysaccharide (<0.8% of Mg only value).

Initial characterization of the P. multocida HAS was performed in thepresence of Mg²⁺. The binding affinity of the enzyme for its sugarnucleotide precursors was assessed by measuring the apparent K_(M)value. Incorporation of [¹⁴C]GlcA or [³H]GlcNAc into polysaccharide wasmonitored at varied concentrations of UDP-GlcNAc or UDP-GlcA,respectively, FIGS. 9 and 10, respectively.

FIG. 9 depicts HAS activity dependence on UDP-GlcNAc concentration.Membranes (20 μg of protein) were incubated with increasingconcentrations of UDP-GlcNAc in buffer containing 50 mM Tris, pH 7, 20mM MgCl₂, and 800 μM UDP-GlcA (1.4×10⁵ dpm of ¹⁴C) for 1 hour. Thebackground radioactivity (identical assay but no added UDP-GlcNAc) wassubtracted from each point. The highest specific incorporation rate intoHA (average approximately 780 dpm/hour) in the titration was defined aV_(max) for normalization to 100%.

FIG. 10 depicts HAS activity dependence on UDP-GlcA concentration. Inexperiments parallel to those described in FIG. 9, increasing amounts ofUDP-GlcA were incubated with 1 mM UDP-GlcNAc (2.7×10⁵ dpm of ³H) underthe same general buffer and assay conditions. The backgroundradioactivity (assay with no added UDP-GlcA) was subtracted from eachpoint. The data is presented as in FIG. 9. Specific incorporation atV_(max) averaged approximately 730 dpm/hour.

In Mg²⁺ containing buffers, the apparent K_(M) values ˜20 μM forUDP-GlcA and ˜75 μM for UDP-GlcNAc were determined utilizing Hanes-Woolfplots ([S]/v versus [S]) of the titration data shown in FIG. 11. FIG. 11depicts the Hanes-Woolf plot estimation of V_(max) and K_(M). Thespecific incorporation data used to generate FIG. 9 (squares) and FIG.10 (circles) were graphed as [S]/v versus [S]. The parallel slopes,which correspond to 1/V_(max), indicate that the maximal velocities forthe sugar nucleotide precursors were equivalent. The x-axis intercept,which signifies −K_(M), yielded K_(M) values of 75 and 20 μM forUDP-GlcNAc and UDP-GlcA, respectively.

The V_(max) values for both sugars were the same because the slopes wereequivalent. In comparison to the results from assays with Mg²⁺, theK_(M) value for UDP-GlcNAc was increased by about 25-50% to ˜105 μM andthe V_(max) increased by a factor of 2-3 fold in the presence of Mn²⁺.These values are represented in Table I. TABLE I membrane wash assay ionK_(M)(μM) V_(max)(pmol/h) Mg Mg 75 ± 5 114 ± 36 EDTA Mg  55 ± 25 98 ± 1EDTA Mn 105 ± 5  380 ± 70

As stated previously, the HA capsules of pathogens P. multocida and S.pyogenes are virulence factors that aid the evasion of host defenses.The HA synthase enzyme from either bacterial source utilizes UDP-sugars,but they possess somewhat different kinetic optima with respect to pHand metal ion dependence and K_(M) values. Both enzymes are most activeat pH 7; however, the PmHAS functions better on the alkaline side of thepH optimum up to at least pH 8.6. On the other hand, the spHASreportedly displays more activity at slightly acidic pH and isrelatively inactive above pH 7.4. The P. multocida enzymes utilizes Mn²⁺more efficiently than Mg²⁺ under the in vitro assay conditions. ThePmHAS binds the UDP-sugars more tightly than streptococcal HasA. Themeasured K_(M) values for the PmHAS in crude membranes are about 2-3fold lower for each substrate than those obtained from the HAS found instreptococcal membranes.

3. Use of the PmHAS for Vaccinations

The DNA sequence of PmHAS may also be used to generate potentialattenuated vaccine strains of P. multocida bacteria after knocking outthe normal microbial gene by homologous recombination with a disruptedversion. Additionally, the PmHAS DNA sequence allows for the generationof diagnostic bacterial typing probes for related P. multocida typesthat are agricultural pathogens of fowl, cattle, sheep and swine.

There are at least five different types of the bacterial pathogen P.multocida with distinct capsule antigens. Fowl cholera or avianpasteurellosis, which is mostly caused by Type A strains, is awidespread, economically damaging disease in commercial poultry. Anacute outbreak of fowl cholera is usually detected only when the birdssuddenly collapse as symptoms often appear just a few hours prior todeath. Although little is known about the molecular basis for thevirulence of P. multocida, apparently one of the pathogen's virulentstrains possesses a polysaccharide capsule, and their colonies display amucoid or“wet” morphology on agar plates. White blood cells havedifficulty engulfing and inactivating the bacteria and the complementcomplex cannot contact the bacterial membrane to cause lysis. The majorcapsule component of the Carter Type A P. multocida, which isresponsible for perhaps 90-95% of fowl cholera disease, is thepolysaccharide HA and HA does not illicit an immune response invirtually all members of the Animal Kingdom. Even if an immune responsedid occur, it would present a problem for the bird because of therepercussions of autoimmune reactions. Type A P. multocida strains arealso prominent causes of swine and bovine pneumatic pasteurellosis, andshipping fever in cattle.

Two other P. multocida capsular types, Type D and Type F, are less wellstudied but are prevalent pathogens in North America. Type F is isolatedfrom about 5-10% of fowl cholera cases. Type D is also a cause ofpneumonia in cattle, sheep, and swine. Isolates from the pneumoniclesions of these domestic animals were analyzed from capsule type andabout 25-40% were Type D and the rest were Type A. Additionally, theType D strain is intimately involved in swine atrophic rhinitis. Thecapsules of these Type D and Type F microbes are composed of differentpolysaccharides with unknown structures, but they are apparently similarto chondroitan, a prevalent molecule on the vertebrate body. The generalbackbone structure of chondroitan, repeating (β1,4)GlcA(β1,3)GalNAcunits, is very similar to HA. It is not surprising, therefore, that theType D and F polysaccharides are poorly immunogenic.

Typically, the antibodies against bacterial surface components derivedfrom previous infections (or vaccinations) are an important means forwhite blood cells to adhere to a bacterium during phagocytosis; thisfeature usually makes the immune response extremely effective infighting disease. Thus, the presence of capsules composed ofnon-immunogenic polymers, such as HA or chondroitan-like sugars,compromise the efficiency of all phases of the host defenses.Streptococcus pyogenes, a human pathogen, also employs a HA capsule asmolecular“mimicry” to protect itself from host defenses. Acapsular S.pyogenes mutants cannot survive in blood and are 100 fold less virulentthan the wild-type in mice. Many virulent E. coli strains possesscapsules comprised of other polysaccharides that mimic host moleculesand aid the cell in eluding the immune system. The capsules of all thesepathogenic bacteria are clever evolutionary adaptations that must beovercome in order to defeat the disease.

Previous investigations have focused on the capsule of P. multocida andits role in virulence. As for Type A fowl strains, wild-typeencapsulated bacteria and various acapsular forms were tested for theirability to survive challenges by isolated host defenses (white bloodcells and complement) or to cause infection and death of live fowl. Theacapsular bacteria were typically: (a) spontaneously arising mutants;(b) chemically-induced mutants; or (c) wild-type bacteria treated withhyaluronidase [HAase], a specific HA-degrading enzyme. In general,capsule-deficient Type A bacteria were more readily killed by isolatedhost defenses in vitro.

Turkey serum killed both mutant and HAase-treated cells, while thewild-type cells multiplied. The complement system was involved, sincethe killing ability was lost by heat or calcium-chelator treatment ofthe serum prior to incubation with bacteria. That encapsulated wild-typecells consume or reduce the level of complement in sera withoutinactivation indicates that the complex binds to but cannot lyse thecells. Turkey macrophages and heterophils phagocytose both acapsularvariants and HAase-treated cells more avidly than wild-type cells.

In live animal testing, the spontaneous mutants were 10³ to 10⁵-foldless virulent than the corresponding wild-type parent strain, asassessed by LD₅₀ (i.e. the lethal does for 50% of the tested animals).This enormous difference shows the importance of the capsule inpathogenesis by Type A strains. The fate of the bacteria in live turkeysalso depended on encapsulation; only wild-type cells survived in theliver. Fifteen to twenty-four hours after injection, wild-type cellswere found in the blood at a 10⁵-fold higher concentration than theunencapsulated mutants. Another role for the HA capsule is adhesion andcolonization. Certain cells in the vertebrate body possess specificHA-binding proteins on their surface; potentially the bacteria couldadhere to the host via this protein/HA interaction.

Likewise, the capsules of Type D and Type F P. multocida are implicatedas virulence factors that confer the microbes with resistance tophagocytosis. When Type D or F cells are treated with chondroitinase,the microbes lose their capsule and are more readily phagocytosed invitro. Furthermore, these polymers are not strongly immunogenic. From invivo testing, encapsulated Type D strains produced more severe nasallesions in swine and had a much lower LD₅₀ in mice than unencapsulatedvariants (10² vs. 10⁷⁻⁸ cells, respectively).

In the case of the Type A and D mutants studied above, however, thegenetic nature of their defects were not known, and there was no facilemethod for mapping the mutations. Particularly with chemicalmutagenesis, there is likely to be more than one mutation in any given“mutant”. Furthermore, it was not shown that HA production wascompletely eradicated in the “acapsular” mutants; thin capsules notdetectable by colony morphology, light microscopy or chemical test couldstill exist. More sensitive radiometric and buoyant density assays arerequired for detection of even small capsules.

Utilizing these new methods, it has been determined that one strain usedin several virulence assays and reported as acapsular, actuallypossesses a very thin HA capsule. Therefore, it is important todetermine the effects from a truly acapsular strain.

Historically, the genes involved in P. multocida capsule production werenot known. Several genes residing in the capsule locus of anotherbacterium in a related genus, Haemophilus influenzae, were mapped andsequenced, but the molecular details of the biosynthetic apparatus arenot available. Even in E. coli, a very well studied Gram-negativeorganism, the exact role of every putative gene product is not wellunderstood, although the loci of capsule formation has been thoroughlymapped and the DNA sequences obtained for several capsule types.

The cloning and sequencing of the HA biosynthesis locus of Streptococcuspyogenes has been reported. This microbe, like P. multocida, utilizes aHA capsule to evade human defenses. The HA operon contains three genesarranged in tandem on an approximately 4 kb of DNA. The first gene,hasA, encodes the 45.1 kDa HA synthase that polymerizes the two sugarnucleotide precursors, UDP-GlcA and UDP-GlcNAc, to form the HApolysaccharide. The second gene, hasB, encodes a 45.5 kDa UDP-glucosedehydrogenase which converts UDP-glucose (UDP-Glc) into UDP-GlcA for HAbiosynthesis. The third gene, hasC, encodes a 34 kDa UDP-glucosepyrophosphorylase which forms UDP-Glc from UTP and glucose-1-phosphate.There is an auxiliary enzyme dedicated to forming UDP-sugars for capsulebiosynthesis; another “housekeeping” gene residing elsewhere in thechromosome supplies UDP-Glc for the bacterium's normal metabolicpathways. UDP-GlcNAc is present in all eubacteria due to its role incell wall synthesis. Therefore, the HA synthase and dehydrogenase arethe only two exogenous proteins needed to direct HA polysaccharidesynthesis in heterologous bacteria. S. pyogenes HA synthase, which ispredicted to be a membrane protein with transmembrane helices, probablyboth polymerizes HA and transports the growing polysaccharide chain tothe outside of the cell.

As previously discussed above, the gene responsible for capsuleproduction of HA in P. multocida, has been isolated and sequenced. (Seee.g., SEQ. ID Nos. 1 and 2.) This gene is disclosed and claimed as partof the present invention. As also discussed previously, the polymers ofthe P. multocida capsules pose a dilemma for host defenses. Using thePmHAS gene sequence information, recombinantly produced P. multocidastrains having the HA synthesis gene “knocked out” will disrupt thebacterial capsular synthesis of P. multocida. Using the “knocked out”strain as a vaccine will allow the host organism to fend off challengesby the pathogen in the field.

As discussed above, Tn916, a versatile and proven mutagen, inserts intothe chromosome of P. multocida at various apparently quasi-randomlocations. The Tn was introduced into the cells on a “suicide”plasmid—i.e. one that cannot replicate in P. multocida—viaelectroporation. The Tn mobilized or jumped off the plasmid and into thegenome at a frequency of about 4,000 events/microgram of DNA. Theresulting progeny possessed the tetracycline resistance gene from Tn916and were easily selectable by the drug.

Also discussed above was the fact that a panel of independent transposonmutants defective in capsule biosynthesis from the virulent parentalstrain were generated. After using a combination of visual andbiochemical screening of the approximately 10⁵ transfected colonies, twoclasses of capsule defects have been found which result in microcapsularor acapsular mutants. The first class (seven independent strains)possessed a very small capsule of HA, hence named microcapsular. Theencapsulated wild-type strain produces large mucoid, iridescent colonieson media plates and the individual cells form a capsule with a thicknessapproximately equal to the cell body diameter as measured by lightmicroscopy. In comparison, the microcapsular strains form smaller,iridescent colonies that appear somewhat drier on media plates; thecapsule thickness of the individual cell is on the order of one quarter(or less) of the wild-type.

Four mutants (four independent strains) appeared to be truly acapsularforming small, dry colonies on media plates. No capsule was detected bylight microscopy. The buoyant density of the acapsular strain, whichdepends on the state of encapsulation, was equivalent to wild-type cellsthat were stripped of their capsule by hyaluronidase treatment. Thesestrains also lacked HA synthase activity; exogenous radiolabeledUDP-sugar precursors were added to the preparations derived from thesemutants but no HA polysaccharide was formed.

Two of the acapsular mutants, TnH and TnL, possessed the interestingproperty that at a frequency of about 10⁻³, occasional revertants with awild-type capsule phenotype appeared on media plates. The revertantcells possessed a wild-type capsule as deemed by light microscopy andradiometric assay for HA polysaccharide. The molecular explanation isthat occasionally the Tn excised neatly from the capsule gene (no addedor deleted bases) and integrates elsewhere in the chromosome. Theresulting encapsulated progeny on media plates are readily observable.This reversion phenomenon is classic genetic proof that the Tn in thesetwo strains was responsible for mutating an important site necessary forcapsule synthesis. However, due to this relative instability, aTn-derived mutant is unsuitable for an attenuated vaccine strain; atsome frequencey, virulent forms could arise.

Southern blotting was used to map the location of the Tn in both theoriginal acapsular mutants and the encapsulated revertants. TnH and TnLhave a Tn element in the same locus as deemed by the pattern afterHindIII or BstXI digestion as shown in FIG. 12. FIG. 12 is a Southernblot mapping of the Tn mutants. Chromosomal DNA from an assortment ofcapsule mutants, encapsulated revertants, and controls were digestedwith HindIII. The DNA was separated by gel electrophoresis and subjectedto Southern blot analysis. The Tn probe recognizes two bands for eachtransposon due to an internal restriction site (forms a large 10 kb anda small 5 kb arm). The Tn probe does not hybridize with DNA from theparental strain without a Tn (lane 0). Multiple DNA preparations derivedfrom separate colonies of the acapsular mutants TnH (H) and TnL (L) orindividual encapsulated revertants (noted with underlined letters) wererun. All the mutants had a single Tn element insertion, except for TnLwhich usually had 2 copies of the Tn (one of the subcultured strains had3 copies). TnW (W) is a mucoid Tn-containing control strain. Thepositions of the lambda HindIII markers (λ) for 23.1, 9.4, and 6.6 kb(top to bottom) are marked.

The Tn element in the acapsular mutants H and L (which have no HAsynthase activity), and a representative microcapsular mutant, TnD (D),map to the same position. Upon reversion to the mucoid phenotype, therelative Tn element moved to a new location in every case. The disruptedDNA from the mutants was isolated at this locus and probes weregenerated for the capsule genes.

Subsequent sequence analysis determined that the TnH and TnL insertionswere approximately 1 kb apart. In all cases, the revertants of thesemutants lost the Tn at the original position and gained a new Tn atdifferent sites (i.e. FIG. 12, lanes with the underlined letters).Alternatively, there has never been an observed reversion of any of themicrocapsular mutants to the encapsulated form. The Tn responsible forall of the microcapsular mutants (typified by TnD) mapped to the same 17kilobase HindIII fragment as the TnH and TnL mutants. Thisco-localization was confirmed by mapping with BstXI as well.

In the other acapsular mutants, TnA and TnG, the Tn elements werelocated in other irrelevant genes and the HA capsule locus was renderednonfunctional by spontaneous mutations. The occasional spontaneousmutation is to be expected; in fact, in similar studies of thestreptococcal capsule locus, 12 out of 13 strains were the result ofspontaneous mutations.

The Tn916 insertional mutagenesis was used to identify and clone the DNAinvolved in HA capsule biosynthesis of P. multocida. In accomplishingthis task, three steps were used: (i) sequencing of host DNA at a Tninsertion site utilizing a primer corresponding to DNA at the terminusof Tn916, (ii) designing PCR primers for the capsule gene based on thenew sequences to amplify the DNA segment between two Tn elements, and(iii) screening wild-type genomic libraries in lambda virus for afunctional clone utilizing the capsule locus-specific PCR product as ahybridization probe.

The key step in obtaining the P. multocida DNA adjacent to the Tn wasthe use of the recently formulated direct sequencing technique which hasbeen fully described in DeAngelis, P. L. (1998) “Transposon Tn916insertional mutagenesis of Pasteurella multocida and direct sequencingof the disruption site,” Microbial Pathogenesis, which is fullyincorporated by reference herein. The P. multocida genome from allcapsular types contains many sites for the restriction enzyme HhaI; thusalmost every DNA fragment in the digest is less than 7 kilobases (kb)and is shown in FIG. 13, lane “O”.

FIG. 13 depicts chimeric DNA templates for sequence analysis of Tndisruption sites. Through this method, the DNA sequence of any geneinterrupted by the Tn916 element can be rapidly and directly obtained.The method capitalizes on the differential sensitivity of the Tn elementand the type A P. multocida genome to the restriction enzyme HhaI. The16 kb Tn element has only one HhaI site resulting in 12 and 4 kbfragments upon digestion. Therefore, any gene interrupted by the Tnelement will have an additional 12 kb of DNA. The increase in HhaIfragment size allows the facile resolution of the Tn-tagged gene fromthe rest of the chromosomal DNA by conventional agarose electrophoresis.This 0.7% gel shows the HhaI digest pattern of chromosomal DNAs from theparental strain without a Tn (lane 0), and several Tn-containingsmutants (Tn mutant lanes). The lambda/HindIII markers (lane S) aredenoted in kb. The chimeric Tn/genomic DNA fragments that migrate atapproximately 13-17 kb (marked with the arrow) are only found in the Tnmutants. Note that lane L has three chimeric bands; this particularmutant has three Tn elements (see FIG. 12).

The chimeric DNA can be isolated and used directly as a sequencingtemplate; no cloning or PCR is required. The resulting large chimericDNA molecule, which is readily separated from the rest of the smallgenomic fragments by agarose gel electrophoresis, serves as the templatein cycle sequencing reactions. A sequencing primer corresponding to theright-hand terminus of the Tn916 directs elongation outward into thedisrupted DNA. Thus, sequence data at the disruption site of mutant DNAcan be routinely obtained without PCR amplification or cloning thetemplate DNA.

The new sequence information was used to design PCR primers foramplification of the region of DNA between the TnL and TnH mutants. Aspecific 1 kb product was used as a hybridization probe to obtain a 5.8kb portion of the capsule biosynthesis operon of Type A P. multocida, asoutlined in FIG. 14 which shows the schematic of HA biosynthesis locusof Type A P. multocida. As FIG. 14 depicts, the insert of a Type Agenomic DNA clone that could direct biosynthesis of HA in E. coli wassequenced. It was found that the open reading frames encode: twoproteins similar to E. coil molecules implicated in polysaccharidetransport, Kps and KfaA; a HA synthase that polymerizes HApolysaccharide; and a precursor forming enzyme, UDP-glucosedehydrogenase. Deletion analysis of the original plasmid showed that anintact HA synthase was essential for HA production in heterologousbacteria. The location of the original Tn insertions corresponding toTnH and TnL are marked with stars. The Tn insertion events apparentlycaused polar mutations that stopped expression of the downstream HAsynthase and dehydrogenase genes. Thus, by using sequence analysis, theintact open reading frames of the novel PmHAS and a putativepolysaccharide transport homolog, similar to E. coli KfaA, were found.The data also shows that an UDP-Glc dehydrogenase homolog, which makesUDP-GlcA precursor, and another transporter protein, an E. coli kps genehomolog, are present near the HA synthase.

The single 110 kDa protein from P. multocida, PmHAS, directs HA capsuleproduction in E. coli. The capsule of the recombinant cells producedwith the PmHAS on a plasmid was as thick as the virulent wild typestrain. The capsular material was deemed authentic HA by itssusceptibility to specific HA lyase digestion and its reactivity withselective HA-binding protein. Interestingly, PmHAS is not very similarto the other HASs at the amino acid level.

In order to make stable isogenic mutants of P. multocida, a modificationof a mutagenesis method employed with P. haemolytica was employed. Aknockout cassette for targeted inactivation by a double crossover of theHA synthase was made. A promoterless chloramphenicol resistance gene(cat) was inserted into the middle of the entire PmHAS open readingframe (at the XhoI site), and cloned into a plasmid (pKK223-3) that doesnot stably replicate in P. multocida. The plasmid was transformed intoencapsulated wild-type strain and the cells were plated on media withchloramphenicol. When integrated into the target gene, the intact PmHASprotein is no longer formed. The cat gene is transcribed by theendogenous capsule gene promoter; the downstream gene, UDP-glucosedehydrogenase should not be affected. Therefore, true isogenic mutantsare formed.

Three isogenic acapsular mutants have been isolated. None of thesemutants were detected as having capsules under light microscopy andIndia ink staining. The HA synthase was disrupted at both the DNA andbiochemical levels. See e.g., Table II. By Western blot analysis usingan antibody directed against a portion of the PmHAS enzyme, theacapsular knockout mutant was missing the approximately 110 KDa band,the PmHAS enzyme, found in the wild-type parent. In combination with thedata in Table II (the lack of polysaccharide production), no functionalPmHAS is found in the knockout strain.

Certain regions are common or very similar between genes of the variouscapsular types. This is shown in the Southern blot analysis of Type D orF DNA depicted in FIG. 15. As depicted in FIG. 15, chromosomal DNA fromType A, F, or D strains was digested with either HindIII or EcoRI (rightor left lane, respectively, for each probe) and subjected to Southernblotting. Digoxigenin-labeled PCR product probes corresponding toregions of either the kfaA homolog (K) or HA synthase (H) genes fromType A were used to detect homologous sequences in bacteria of othercapsule types (bands marked with stars). KfaA homologs are apparent inboth Type F and D. A very similar synthase homolog is found in Type F,but not in Type D. The probes are suitable for screening libraries. Thelambda/HindIII standards are marked in kilobases.

Type F has regions that are similar to both probes, while Type D wasonly similar to the transporter protein probe. PCR was used with severalsets of primers corresponding to the Type A sequences to amplify Type Dor F genomic DNA as shown in FIG. 16. PCR of heterologous DNA with TypeA primers is depicted in FIG. 16. Various primer pairs corresponding tothe kfaA homolog gene (Panel A) or the HA synthase gene (Panel B) of theType A strain were used to amplify genomic DNA isolated from severalother P. multocida strains with different capsule types. Forty cycles(94° C., 30 sec; 42° C., 30 sec; 72° C., 60 sec) of polymerase chainreaction with Taq enzyme were performed.

The reaction mixes were separated on a 10/0 agarose gel and stained withethidium (lanes: A, Type A; D, Type D; F, Type F; 0, no templatecontrol. Standards (S) are the 100 bp ladder, the 1 and 0.5 kb bands aremarked with arrows. The P-I primer pair shows products for all threecapsule types, but the Type D product is smaller than the otherproducts. The P-II and P-III pairs amplify Type A and F DNA only. Incontrast, the P-IV and P-V pairs amplify Type A only. It appears thatthe Type A and F capsule loci are more similar to each other than totype D. The PCR products from the P-I primer pair will serve as goodhybridization probes for the capsule locus from other types.

Not all combinations of primers yielded PCR products with theheterologous template DNA. 0.2-1 kb portions of Type F genes encodingthe HA synthase or the capsule polysaccharide transporter analog wereamplified. Also amplified was a 1 kb region of the Type D genomeencoding the transporter protein. Sequence analysis of several PCRproducts revealed homologous yet distinct sequences. Overall, this datasuggests that the Type A and F strains are more related to each otherand not as similar to type D. Sequence comparison of Type A and F KfaAhomologs and E. coli KfaA is shown in FIG. 17. The PCR product that wasgenerated by amplification of Type F DNA with the P-I primer set (seeFIG. 16) was gel-purified and sequenced with one of the originalprimers. It was found that the type A and F sequences were very similarat the amino acid level; this partial alignment of the protein sequencesshows that in this region the sequences are largely identical with somemismatches (the differences are underlined in FIG. 17). Overall, the P.multocida sequences are quite homologous to the E. coli KfaA protein,which is implicated in polysaccharide transport (the identical residuesare bolded in FIG. 17). These PCR products will also be useful ashybridization probes to obtain functional capsule loci from Type D or Fgenomic libraries. The cloned DNA also allows the construction of geneknockout plasmids: wherein, the resulting mutant strains are useful forvirulence assays or vaccines.

The production of the bacterial capsule of P. multocida involves atleast the following steps: (i) synthesis of sugar nucleotide precursors;(ii) polymerization of precursors to form the capsular polysaccharide;and (iii) export or transport of the polysaccharide to the extracellularspace where capsule assembly occurs. Of course, there are potentialregulatory genes or factors that control enzyme levels or enzymaticactivity, but the focus is on the major structural enzymes of thepathway. In E. coli, the candidate type 2 capsule genes encoding enzymesfor this process are located together at a single site on the bacterialchromosome. E. coli strains that make capsules with different structureshave varied enzymes for step (i) and (ii) above, but all appear to sharea common transport/export machinery for step (iii).

It has been discovered that in S. pyogenes, a single integral membraneenzyme polymerizes the precursor sugars, and also transports the HApolysaccharide across the membrane. Type A P. multocida has fourdifferent genes that are involved in each of the three biosyntheticsteps for bacterial capsule production. (See FIG. 14) The similarity ofthe P. multocida polysaccharide transporter to the E. coli homolog atthe protein level suggests that the general functions of some othercapsule genes may also be similar to these two species.

The role of the capsule as a virulence factor in fowl cholera has beenassessed. In order to avoid pitfalls and caveats encountered in studiesof bacterial capsules and virulence, defined mutants were compared tothe wild-type microbes. Isogenic Type A mutants having disrupted capsulegenes were tested for their ability to avoid preexisting or preimmunehost defenses in vitro, as well as to infect living fowl in vivo. Thestable isogenic mutants were produced according to the methods describedhereinabove. Using a disrupted version of the PmHAS gene on a plasmid(see FIG. 18) and homologous recombination, a recombinant P. multocidastrain was created that had lost the ability to make a hyaluronancapsule. The strain was further analyzed at both the DNA and biochemicallevels. We found that the functional HA synthase gene was replaced witha defective gene containing a cat cassette disruption by both Southernblot and PCR analyses. (See FIG. 19).

Confirmation of gene disruption is shown in FIG. 19. Panel A is aSouthern blot analysis. Chromosomal DNA from various strains wasdigested with HindIII, separated on a 0.7% agarose gel, and transferredto nitrocellulose. The blot was hybridized with a P. multocida HAS geneprobe. Two bands were detected due to an internal HindIII restrictionsite in PmHAS gene. Lane M is the mucoid transformant; Lane KO is theacapsular knockout mutant; Lane P is the parental strain. The additionof the 670 bp cat cassette causes the size shift of the upper band inthe KO lane (marked with an arrow).

Panel B of FIG. 19 is a PCR analysis. The DNA in cell lysates fromvarious strains was amplified by 35 cycles of PCR with a pair ofoligonucleotide primers that flank the XhoI site of PmHAS. The length ofthe amplicon from the normal, wild-type gene is 650 base pairs. The PCRreactions were separated on a 1% agarose gel and visualized withethidium bromide. Lane M is the mucoid transformant; Lane KO is theacapsular knockout mutant; Lane P is the parental strain; Lane C is thecloned PmHAS plasmid control; and Lane S are the size standards. The PCRproduct produced by the knockout mutant template is approximately 1,300bp (marked with arrow); this band is composed of the 670 bp cat cassetteand the 650 bp derived from PmHAS. No wild-type amplicon is detected inthe knockout strain reaction, therefore, homologous recombinationmediated by a double crossover event occurred.

Furthermore, utilizing a sensitive radiochemical assay for HApolysaccharide, it was found that the mutant strain did not produce HA,and is shown in Table II which lists the HA production of variousstrains. TABLE II HA polysaccharide Strain (nanograms/ml per OD₆₀₀) P =wild type parent 1,200 M = Mucoid transformant 1,200 KO = Acapsularknockout mutant ≦0.05The strains listed in Table II were overnight cultures of the variousstrains which were tested for the presence of HA polysaccharide usingthe specific radiometric assay outlined hereinabove. The cultures werenormalized by spectrophotometry and the data was presented as theconcentration of HA in a culture with an absorbance of 1.0 at 600 nm.The wild-type parent or a mucoid, encapsulated transformant synthesizedsubstantial amounts of HA. In contrast, no detectable HA was produced bythe acapsular knockout mutant (KO). Thus, the role of the capsule invirulence could be assessed. The methodology employed could also be usedto construct other mutants of P. multocida and one of ordinary skill inthe art, given the disclosure of the present invention, could accomplishsuch a task.

Animal testing has compared the in vivo pathogenicity of the mutants tocomplemented mutant controls and wild-type Type A P. multocida. Theknockout strain of Type A Pasteurella multocida ATCC 15742, (whichcauses fowl cholera), was shipped to the USDA Research Station in Ames,Iowa for virulence testing. Using targeted homologous recombination,capsule biosynthesis of the knockout strain has been disrupted and theknockout strain was predicted to be 1,000-fold less virulent.

The virulence testing was carried out to check the safety of the KOstrain as a vaccine strain. Turkey eggs were hatched in clean conditionsand-raised to the age of two weeks. The poults were injected withvarious concentrations of bacteria (either wild-type parent or theknockout strain). The bacterial count was enumerated by spectroscopy andcolony counting after plating. The animals were injected intramuscularlyand placed in a biological containment pen. The inoculated poults(groups of 6 or 7 per microbial dose ranging from about 80 to 10⁷bacteria in 10 fold steps)were observed. The general appearance, levelof activity and morbidity was checked for 6 days. Dead or dying birdswere autopsied and checked for the presence of lesions, abscesses, andorgan failure.

The results of the in vivo experiments are summarized in Table III.TABLE III Strain No. of cells per injection Mortality rate Wild-typewild type 8 × 10³ 43% Wild-type wild type 860 17% Mutant w/HAS knockout1 × 10⁷  0%The point of this type of testing was to assess the general trends ofinfection with respect to encapsulation of the pathogen. For eachdetermination, white turkeys were inoculated with a titered amount ofbacteria IM. Symptoms and death of the turkeys was measured andtabulated in order to compare the relative virulence of the mutants.Protection trials will be conducted in order to determine if immunizedturkeys can survive a challenge with wild-type virulent organisms.

Type A knockout strains that infect cattle and rabbits have also beenprepared. Testing will be conducted in vivo in order to determine boththe pathogenicity of these knockout strains as well as protection trialsto determine if the immunized animals can survive a challenge withwild-type virulent organisms.

Two main types of protection experiments will be performed. First,passive immunization is done. One chicken is infected with the potentialvaccine KO strain and a sample of its serum (with protective antibodies)is taken about 1-2 weeks after inoculation. This sera or derivedpurified antibody is injected into a naive chicken. The naive chicken ischallenged with wild-type strain. The bird, if it receives protectiveantibody, will survive the challenge with the otherwise lethal wild-typebacteria.

Second, active immunization will be undertaken. In this case, the samechicken is sequentially infected with the potential vaccine KO strain,and a few weeks later, the bird is challenged with a normally lethaldose of wild-type bacteria. In this case antibody-mediated and cellmediated immunity are tested.

Using the present invention, it is predicted that there are similaritiesin the capsule loci of the various encapsulated types of P. multocidabecause of the close structural similarity of the polysaccharides. Thepresent invention also relates to a homologous Type F P. multocida gene(“PmCS”). The PmCS sequence information is provided in SEQ ID NO:3. TheType F gene is approximately 85% identical to the Type A gene and thesequence comparison is shown in FIG. 20. This homology was found at theDNA level between the cloned type A capsule genes and certain regions ofthe Type D and F genomes by Southern blotting and PCR, as shown in FIGS.15, 16, and 17. Libraries of Type F genomic DNA in lambda phage werescreened to isolate the homologous capsule loci. Libraries of Type Dgenomic DNA in lambda phage will be screened to isolate the homologouscapsule loci and one of ordinary skill in the art would appreciate andunderstand that the Type D capsule loci can be determined in exactly thesame manner as with the Type A and F. The type A and F PmHAS sequencesare 89% similar.

The Type F polysaccharide synthase gene was obtained by using a PCRproduct hybridization probe, FIG. 16, joining the HAS homolog and theKfa homolog. A 3 kb amplicon was produced using genomic DNA from a TypeF strain and the appropriate primers from a Kfa and synthase regions.This material was labeled with digoxigenin and used to obtain a cloneand subsequently a plasmid from a Type F genomic DNA library in LambdaZAP Express library (as described for the Type A cloning. The positivelyhybridizing clone was sequenced. As in the case of the Type A HAsynthase gene, PmHAS, the functionality was checked by expression in thepKK223-3 (Pharmacia) vector in E. coli. It was found that this enzymeincorporated in vitro UDP-GalNAc and UDP-GlcA into high molecular weightpolymer as expected for a chondroitin molecule.

The capsular polysaccharide synthases were monitored with antibodies andWestern blot analysis. The antibodies were generated against a syntheticpeptide that corresponds to a shared, homologous region (12-20 aminoacid residues) of the synthase enzymes. Western blots confirmed thatboth Type A and Type F P. multocida had an immunoreactive 110 kDaprotein by SDS-PAGE.

FIG. 21 is a Western blot analysis of native and recombinant PmHASproteins. The native PmHAS and various recombinant truncatedPmHAS-derived proteins made in E. coli were compared on SDS-PAGE gels.For the recombinant samples, the total lysate (T), the membranes (M),and the cytoplasm (C) were subjected to Western blotting with ananti-PmHAS antibody. The original protein found in native Pasteurellamultocida (Pm, lane W; marked with an arrow) migrates at about 110 kDa;the knockout vaccine strain (KO lane) is missing this band. The nativePmHAS and a recombinant version missing a portion of the carboxylterminus (PmΔCC) had HA synthase activity. The other truncatedconstructs were inactive.

4. Use of the PmHAS in Diagnostic Applications

The present invention also relates to the generation of useful probesthat facilitate the identification of Type A, D, and F P. multocida orP. haemolytica in the field. The diagnosis of which particular strain ispresent in animals is currently determined by serology, agglutination,or DNA fingerprinting after restriction analysis. The former two methodscan be problematic, frequently yield false identification, and varydepending on the source of typing antiserum. Capsular serology of theCarter Types A, D, and F does not even employ an antibody because thesepolymers are such poor immunogens. Instead, laborious assays involvingenzymatic digestion or cell flocculation with acriflavine are routinelyemployed. DNA fingerprinting is accurate, but it relies on extensiveknowledge of numerous type strains on file. Sets of capsule-specificprimers will be used to readily perform these epidemiological studies,specifically by using rapid, facile PCR analysis to identify pathogenicisolates in half a day with minimal handling and no subculturing. Oncethe pathogen is identified, a more informed decision could be made onthe choice of antibiotic or vaccine.

The utility of the use of capsule DNA information to quickly ascertainthe type of P. multocida is obvious in light of the problems withcurrent typing methods. Either hybridization or PCR-based typing isenvisioned as practical, sensitive, and rapid. One specific embodimentwould be to bring an appropriately labeled or tagged synthase DNA probe(or by extension a capsule locus gene which differs among capsule type)that by virtue of its uniqueness can be distinguished under appropriatehybridization conditions (e.g. complementary gene and probes hybridizeto yield a signal while nonidentical gene from another capsular typedoes not hybridize thus no signal is obtained). Another specificembodiment would be to design PCR primers that can distinguish thecapsule types. An amplicon of the correct size would signify aparticular capsule type; no amplicon signifies another distinct capsuletype.

In the current state of the art, several PCR primer pairs which givedistinguishable and different size bands in a single reaction can beenvisioned. Such a multiplex method would allow many reactions to beperformed simultaneously. The knowledge of the DNA sequence of thevarious capsule biosynthesis loci, in particular the synthases, allowsthese tests to rapidly distinguish the various pathogenic strains.

Thus, it should be apparent that there has been provided in accordancewith the present invention an isolated and sequenced PmHAS and a methodsfor making and using the PmHAS and knockout strains of P. multocida thatfully satisfy the objectives and advantages set forth above. Althoughthe invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

1. A purified and isolated nucleic acid sequence encoding enzymaticallyactive hyaluronan synthase, the nucleic acid sequence selected from thegroup consisting of: (a) the nucleic acid sequence as set forth in SEQID NO:2; and (b) a nucleic acid sequence encoding an enzymaticallyactive hyaluronan synthase, wherein a complement of the nucleic acidsequence is capable of hybridizing to the nucleic acid sequence of SEQID NO:2 under standard hybridization conditions.
 2. A procaryotic oreucaryotic host cell transformed or transfected with a nucleic acidsegment according to claim 1 in a manner allowing the host cell toexpress hyaluronic acid.
 3. An isolated nucleic acid segment consistingessentially of a nucleic acid segment encoding enzymatically activehyaluronan synthase from Pasteurella, wherein a complement of thenucleic acid sequence is capable of hybridizing to the nucleic acidsequence of SEQ ID NO:2 under standard hybridization conditions.
 4. AcDNA sequence according to claim
 3. 5. A procaryotic or eucaryotic hostcell transformed or transfected with a nucleic acid segment according toclaim 4 in a manner allowing the host cell to express hyaluronic acid.6. An isolated nucleic acid segment consisting essentially of a nucleicacid segment encoding enzymatically active hyaluronan synthase fromPasteurella, wherein the nucleic acid segment is essentially as setforth in SEQ ID NO:2.
 7. A cDNA sequence according to claim
 6. 8. Aprocaryotic or eucaryotic host cell transformed or transfected with anucleic acid segment according to claim 6 in a manner allowing the hostcell to express hyaluronic acid.
 9. An isolated nucleic acid segmentconsisting essentially of a nucleic acid segment encoding enzymaticallyactive hyaluronan synthase from Pasteurella, wherein the nucleic acidsegment is at least 85% identical to SEQ ID NO:2.
 10. A cDNA sequenceaccording to claim
 9. 11. A procaryotic or eucaryotic host celltransformed or transfected with a nucleic acid segment according toclaim 9 in a manner allowing the host cell to express hyaluronic acid.12. An isolated nucleic acid segment consisting essentially of a nucleicacid segment encoding enzymatically active hyaluronan synthase fromPasteurella, wherein the nucleic acid segment is essentially as setforth in SEQ ID NO:2 and encodes a hyaluronan synthase, wherein thehyaluronan synthase has SEQ ID NO:18 and DSD motifs therein.
 13. A cDNAsequence according to claim
 12. 14. A procaryotic or eucaryotic hostcell transformed or transfected with a nucleic acid segment according toclaim 12 in a manner allowing the host cell to express hyaluronic acid.15. An isolated nucleic acid segment consisting essentially of a nucleicacid segment encoding enzymatically active hyaluronan synthase fromPasteurella, wherein the hyaluronate synthase encoded by the nucleicacid segment is at least 89% identical to SEQ ID NO:1.
 16. A cDNAsequence according to claim
 15. 17. A procaryotic or eucaryotic hostcell transformed or transfected with a nucleic acid segment according toclaim 15 in a manner allowing the host cell to express hyaluronic acid.